Maize RS81 promoter and methods for use thereof

ABSTRACT

The current invention provides the maize RS81 promoter. Compositions comprising this sequence are described, as are plants transformed with such compositions. Further provided are methods for the expression of transgenes in plants comprising the use of these sequences. The methods of the invention include the direct creation of transgenic plants with the RS81 promoter by genetic transformation, as well as by plant breeding methods. The sequences of the invention represent a valuable new tool for the creation of transgenic plants, preferably having one or more added beneficial characteristics.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to transgenic plants. Morespecifically, it relates to methods and compositions for transgeneexpression using the maize RS81 promoter.

2. Description of the Related Art

An important aspect in the production of genetically engineered crops isobtaining sufficient levels of transgene expression in the appropriateplant tissues. In this respect, the selection of promoters for directingexpression of a given transgene is crucial. Promoters which are usefulfor plant transgene expression include those that are inducible, viral,synthetic, constitutive as described (Poszkowski et al., 1989; Odell etal., 1985), temporally regulated, spatially regulated, andspatio-temporally regulated (Chau et al., 1989).

A number of plant promoters have been described with various expressioncharacteristics. Examples of some constitutive promoters which have beendescribed include the rice actin 1 (Wang et al., 1992; U.S. Pat. No.5,641,876), CaMV 35S (Odell et al., 1985), CaMV 19S (Lawton et al.,1987), nos (Ebert et al., 1987), Adh (Walker et al., 1987), sucrosesynthase (Yang & Russell, 1990).

Examples of tissue specific promoters which have been described includethe lectin (Vodkin et al., 1983; Lindstrom et al., 1990), corn alcoholdehydrogenase 1 (Vogel et al., 1989; Dennis et al., 1984), corn lightharvesting complex (Simpson, 1986; Bansal et al., 1992), corn heat shockprotein (Odell et al., 1985; Rochester et al., 1986), pea small subunitRuBP carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), Tiplasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopalinesynthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunenet al., 1988), bean glycine rich protein 1 (Keller et al., 1989),truncated CaMV 35s (Odell et al., 1985), potato patatin (Wenzler et al.,1989), root cell (Conkling et al., 1990), maize zein (Reina et al.,1990; Kriz et al., 1987; Wandelt and Feix, 1989; Langridge and Feix,1983; Reina et al., 1990), globulin-1 (Belanger and Kriz et al., 1991),α-tubulin, cab (Sullivan et al., 1989), PEPCase (Hudspeth & Grula,1989), R gene complex-associated promoters (Chandler et al., 1989), andchalcone synthase promoters (Franken et al., 1991).

Inducible promoters which have been described include ABA- andturgor-inducible promoters, the promoter of the auxin-binding proteingene (Schwob et al., 1993), the UDP glucose flavonoidglycosyl-transferase gene promoter (Ralston et al., 1988); the MPIproteinase inhibitor promoter (Cordero et al., 1994), and theglyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al.,1995; Quigley et al., 1989; Martinez et al., 1989).

A class of genes which are expressed in an inducible manner areglycine-rich proteins. Expression of glycine rich proteins is induced bythe plant hormone abscibic acid (ABA). Genes encoding glycine richproteins have been described, for example, from maize (Didierjean etal., 1992; Gomez et al., 1988; Baysdorfer, Genbank Accession No.AF034945) sorghum (Cretin and Puigdomenech, 1990), and rice (Lee et al.,Genbank Accession No. AF009411).

In addition to the use of a particular promoter, expression oftransgenes can be influenced by other types of elements. In particular,introns have demonstrated the potential for enhancing transgeneexpression. For example, Callis et al. (1987) described an intron fromthe corn alcohol dehydrogenase gene which is capable of enhancing theexpression of transgenes in transgenic plant cells. Similarly, Vasil etal. (1989) described an intron from the corn sucrose synthase genehaving similar enhancing activity. The rice actin 1 intron, inparticular, has found wide use in the enhancement of transgeneexpression in a number of different transgenic crops (McElroy et al.,1991). This 5′ intron was identified from the first coding exon of therice actin 1 sequence (McElroy et al., 1990). Plant actin is encoded bya gene family present in all plant species studied to date (Meagher,1991). In rice, there are at least eight actin-like sequences perhaploid genome. Four of the rice actin coding sequences (rice actin 1,2, 3 and 7) have been isolated and shown to differ from each other inthe tissue and stage-specific abundance of their respective transcripts(Reece, 1988; McElroy et al., 1990a; Reece et al., 1990; U.S. Pat. No.5,641,876; Genbank Accession numbers X15865, X15864, X15862, and X15863,respectively).

While the above studies have provided a number of useful tools for thegeneration of transgenic plants, there is still a great need in the artfor novel promoter and enhancer sequences with beneficial expressioncharacteristics. In particular, there is a need in the art forpromoter-enhancer combinations which are capable of directing high-levelexpression of exogenous genes in transgenic crop plants. Many previouslyidentified regulatory sequences fail to provide the levels of expressionrequired to fully realize the benefits potentially conferred byexpression of selected genes in transgenic plants. Additionally, manyregulatory regions fail to demonstrate suitable or desirable expressionprofiles for transgene expression. There is, therefore, a great need inthe art for the identification of novel sequences which can be used forthe high-level expression of selected transgenes in economicallyimportant crop plants.

SUMMARY OF THE INVENTION

In one aspect, the invention provides an isolated nucleic acidcomprising a maize RS81 promoter. Also provided by the invention is amaize RS81 promoter isolatable from the nucleic acid sequence of SEQ IDNO:1. The RS81 promoter may, in particular embodiments of the invention,comprise from about 50 to about 2584, from about 150 to about 2584, fromabout 250 to about 2584, from about 400 to about 2584, from about 750 toabout 2584, from about 1000 to about 2584, from about 1500 to about2584, or from about 2000 to about 2584, contiguous nucleotides of thenucleic acid sequence of SEQ ID NO:1. The maize RS81 promoter providedby the invention may further comprise the nucleic acid sequence of SEQID NO:1. In particular embodiments of the invention, the maize RS81promoter may further comprise an enhancer, including an intron such asthe rice actin 1 intron or the rice actin 2 intron. An RS81 promoterused in accordance with the invention may further include a terminator,for example, an rbcS terminator.

In another aspect, the invention provides an expression cassettecomprising a maize RS81 promoter operably linked to a selected DNA.Potentially any selected DNA may be included with the expressioncassette, including those encoding an insect resistance protein, abacterial disease resistance protein, a fungal disease resistanceprotein, a viral disease resistance protein, a nematode diseaseresistance protein, a herbicide resistance protein, a protein affectinggrain composition or quality, a nutrient utilization protein, amycotoxin reduction protein, a male sterility protein, a selectablemarker protein, a screenable marker protein, a negative selectablemarker protein, an environment or stress resistance protein, or aprotein affecting plant agronomic characteristics. The expressioncassette may further include any suitable selectable marker protein.Examples of suitable selectable marker proteins include phosphinothricinacetyltransferase, glyphosate resistant EPSPS, aminoglycosidephosphotransferase, hygromycin phosphotransferase, dalapon dehalogenase,bromoxynil resistant nitrilase, anthranilate synthase and glyphosateoxidoreductase.

An expression cassette may further comprise an enhancer. In particularembodiments of the invention, the enhancer is selected from the groupconsisting of the rice actin 1 intron and the rice actin 2 intron. Theexpression cassette may still further comprise a transit peptide codingsequence, for example, a transit peptide selected from the groupconsisting of chlorophyll a/b binding protein transit peptide, smallsubunit of ribulose bisphosphate carboxylase transit peptide, EPSPStransit peptide and dihydrodipocolinic acid synthase transit peptide. Inthe expression cassette, the selected coding sequence may be operablylinked to potentially any terminator, for example, a rice or other typeof rbcS terminator.

In yet another aspect, the invention provides an expression vectorcomprising a maize RS81 promoter operably linked to a selected codingregion. In one embodiment of the invention, the expression vector may befurther defined as a plasmid vector. In another embodiment of theinvention, the plasmid vector may be located within a bacterial cell.

In still yet another aspect, the invention provides a fertile transgenicplant which is stably transformed with a selected DNA comprising a maizeRS81 promoter. In particular embodiments of the invention, the fertiletransgenic plant comprises a maize RS81 promoter which is isolatablefrom the nucleic acid sequence of SEQ ID NO:1. In other embodiments ofthe invention, the fertile transgenic plant has a maize RS81 promotercomprising from about 50 to about 2584, from about 100 to about 2584,from about 200 to about 2584, from about 400 to about 2584, from about750 to about 2584, from about 1000 to about 2584, from about 1500 toabout 2584, or from about 2000 to about 2584, contiguous nucleotides ofthe nucleic acid sequence of SEQ ID NO:1. The fertile transgenic plantmay also comprise the nucleic acid sequence of SEQ ID NO:1. In thefertile transgenic plant, the selected DNA may further comprise aselected coding region operably linked to said maize RS81 promoter.

The selected coding region may be potentially any protein, for example,an insect resistance protein, a bacterial disease resistance protein, afungal disease resistance protein, a viral disease resistance protein, anematode disease resistance protein, a herbicide resistance protein, aprotein affecting grain composition or quality, a nutrient utilizationprotein, an environment or stress resistance protein, a mycotoxinreduction protein, a male sterility protein, a selectable markerprotein, a screenable marker protein, a negative selectable markerprotein, or a protein affecting plant agronomic characteristics.

Where a selected protein used in accordance with the invention is aselectable marker protein, it may be preferable to utilize a proteinselected from the group consisting of phosphinothricinacetyltransferase, glyphosate resistant EPSPS, aminoglycosidephosphotransferase, hygromycin phosphotransferase, neomycinphosphotransferase, dalapon dehalogenase, bromoxynil resistantnitrilase, anthranilate synthase and glyophosate oxidoreductase. Infurther embodiments of the invention, the selected coding region isoperably linked to a terminator, for example, a rice or other rbcSterminator. The selected DNA may also comprise an enhancer, such as arice actin 1 intron or rice actin 2 intron. The selected DNA maycomprise plasmid DNA and/or a transit peptide. In particular embodimentsof the invention, the transit peptide is selected from the groupconsisting of chlorophyll a/b binding protein transit peptide, smallsubunit of ribulose bisphosphate carboxylase transit peptide, EPSPStransit peptide and dihydrodipocolinic acid synthase transit peptide.

In one embodiment of the invention, a fertile transgenic plant preparedin accordance with the invention is further defined as amonocotyledonous plant, for example, a monocot selected from the groupconsisting of wheat, maize, rye, rice, oat, barley, turfgrass, sorghum,millet and sugarcane. The fertile transgenic plant also may be adicotyledonous plant, for example, a tobacco, tomato, potato, soybean,cotton, canola, alfalfa or sunflower plant. In one embodiment of theinvention, the plant is a soybean plant.

In still yet another aspect, the invention provides a fertile R₀transgenic plant comprising a transgenic RS81 promoter. Also providedare seeds of the R₀ transgenic plant, wherein the seeds comprise aselected DNA including the RS81 promoter. Further provided are progenyplants of any generation of the R₀ transgenic plant, wherein said R₀transgenic plant comprises said selected DNA, as well as seeds of theprogeny plants, wherein said seed comprises said selected DNA.

In still yet another aspect, the invention provides a crossed fertiletransgenic plant prepared according to the method comprising the stepsof: (i) obtaining a fertile transgenic plant comprising a selected DNAcomprising a maize RS81 promoter; (ii) crossing said fertile transgenicplant with itself or with a second plant lacking said selected DNA toprepare the seed of a crossed fertile transgenic plant, wherein saidseed comprises said selected DNA; and (iii) planting said seed to obtaina crossed fertile transgenic plant. The invention also provides a seedof the crossed fertile transgenic plant, wherein said seed comprisessaid selected DNA. In one embodiment of the invention the crossedfertile transgenic plant is a monocotyledonous plant, including a wheat,oat, barley, maize, rye, rice, turfgrass, sorghum, millet or sugarcaneplant. The crossed fertile transgenic plant may also be a dicotyledonousplant, including a tobacco, tomato, potato, soybean, canola, alfalfa,sunflower or cotton plant. The selected DNA may be inherited through amale or female parent. In one embodiment of the invention, the secondplant is an inbred plant, and the crossed fertile transgenic plant is ahybrid. In particular embodiments of the invention, said maize RS81promoter is isolatable from the nucleic acid sequence of SEQ ID NO:1. Infurther embodiments of the invention, the maize RS81 promoter comprisesfrom about 50 to about 2584, from about 100 to about 2584, from about200 to about 2584, from about 400 to about 2584, from about 750 to about2584, from about 1000 to about 2584, from about 1500 to about 2584, orfrom about 2000 to about 2584 contiguous nucleotides of the nucleic acidsequence of SEQ ID NO:1. The maize RS81 promoter may also comprise thenucleic acid sequence of SEQ ID NO:1.

The crossed fertile transgenic plant may comprise a selected DNAcomprising a selected coding region operably linked to theaforementioned maize RS81 promoter. In particular embodiments of theinvention, the selected protein is selected from the group consisting ofinsect resistance protein, a bacterial disease resistance protein, afungal disease resistance protein, a viral disease resistance protein, anematode disease resistance protein, a herbicide resistance protein, aprotein affecting grain composition or quality, a nutrient utilizationprotein, a mycotoxin reduction protein, a male sterility protein, aselectable marker protein, a screenable marker protein, a negativeselectable marker protein, a protein affecting plant agronomiccharacteristics, and an environment or stress resistance protein. Theselected DNA may also comprise an enhancer, such as a rice actin 1intron or rice actin 2 intron. The selected protein may be operablylinked to a terminator, for example, a rice or other rbcS terminator.

In still yet another aspect, the invention provides a method ofexpressing a selected protein in a transgenic plant comprising the stepsof: (i) obtaining a construct comprising a selected coding regionoperably linked to a maize RS81 promoter; (ii) transforming a recipientplant cell with said construct; and (iii) regenerating a transgenicplant expressing said selected protein from said recipient plant cell.In one embodiment of the invention, the step of transforming comprises amethod selected from the group consisting of microprojectilebombardment, PEG mediated transformation of protoplasts,electroporation, silicon carbide fiber mediated transformation, orAgrobacterium-mediated transformation. In another embodiment therecipient plant cell is from a monocotyledonous plant, including awheat, maize, rye, rice, turfgrass, oat, barley, sorghum, millet, orsugarcane plant. In further embodiments, the recipient plant cell isfrom a dicotyledonous plant, including a tobacco, tomato, potato,soybean, canola, sunflower, alfalfa or cotton plant. The selectedprotein can be any protein, for example, an insect resistance protein, abacterial disease resistance protein, a fungal disease resistanceprotein, a viral disease resistance protein, a nematode diseaseresistance protein, a herbicide resistance protein, a protein affectinggrain composition or quality, a nutrient utilization gene, a mycotoxinreduction protein, a male sterility protein, a selectable markerprotein, a screenable marker protein, a negative selectable marker gene,a gene affecting plant agronomic characteristics, or an environment orstress resistance protein. The construct also may comprise an enhancer,for example, a rice actin 1 intron or rice actin 2 intron. The selectedcoding region also may be operably linked to a terminator, for example,a rice or other rbcS terminator.

In still yet another aspect, the invention provides a method of plantbreeding comprising the steps of: (i) obtaining a transgenic plantcomprising a selected DNA comprising a maize RS81 promoter; and (ii)crossing said transgenic plant with itself or a second plant. In oneembodiment of the invention, the transgenic plant is a monocotyledonousplant, for example, a wheat, maize, oat, barley, rye, rice, turfgrass,sorghum, millet or sugarcane plant. In another embodiment of theinvention, the transgenic plant is a dicotyledonous plant, for example,a tobacco, tomato, potato, soybean, canola, sunflower, alfalfa or cottonplant. In still another embodiment of the invention, the maize RS81promoter is isolatable from the nucleic acid sequence of SEQ ID NO:1.The maize RS81 promoter may also comprise from about 100 to about 2584,from about 150 to about 2584, from about 250 to about 2584, from about400 to about 2584, from about 600 to about 2584, from about 800 to about2584, from about 1000 to about 2584, from about 1500 to about 2584, orfrom about 2000 to about 2584 contiguous nucleotides of the nucleic acidsequence of SEQ ID NO: 1. The maize RS81 promoter may also comprise thenucleic acid sequence of SEQ ID NO:1.

In the method of plant breeding, the transgenic plant may be crossedwith said second plant, and said second plant may be an inbred plant.The method may also further comprise the steps of: (iii) collectingseeds resulting from said crossing; (iv) growing said seeds to produceprogeny plants; (v) identifying a progeny plant comprising said selectedDNA; and (vi) crossing said progeny plant with itself or a third plant.In the method, said progeny plant may inherit the selected DNA through amale or female parent. In one embodiment of the invention, the secondplant and said third plant are of the same genotype, and may be inbredplants.

The selected DNA may further comprise potentially any protein, includingan insect resistance protein, a bacterial disease resistance protein, afungal disease resistance protein, a viral disease resistance protein, anematode disease resistance protein, a herbicide resistance protein, aprotein affecting grain composition or quality, a nutrient utilizationprotein, a mycotoxin reduction protein, an environment or stressresistance protein, a male sterility protein, a selectable markerprotein, a screenable marker protein, a negative selectable markerprotein, or a protein affecting plant agronomic characteristics. Theselected DNA may further comprise a genetic element which enhances theexpression of said protein in said transgenic plant, for example, a riceactin 1 intron or rice actin 2 intron.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Sequence of the 5′ region of the maize RS81 gene. (SEQ ID NO:1)

FIG. 2: Sequence of the 3′ end of the rice rbcS sequence (SEQ ID NO:2).Transcribed nucleotides are underlined. The polyadenylation signal isgiven by bold nucleotides.

FIG. 3: Sequence of the 5′ region of the rice actin 2 gene (SEQ IDNO:3). The actin 2 intron (SEQ ID NO:4) is indicated by lowercaseitalicized nucleotides, uppercase nucleotides indicate the actin 2 exon1, lower case nucleotides indicate the actin 2 promoter, upper caseitalics indicate the actin 2 exon 2, and upper case bold italicsindicate the actin 2 translation initiation codon.

FIG. 4A, 4B: Histological detection of RS81-directed GUS expression inroot endodermis. FIG. 4A: Safranin O counterstaining of root tissue;FIG. 4B: Histological detection of GUS expression.

DETAILED DESCRIPTION OF THE INVENTION

The current invention overcomes deficiencies in the prior art byproviding novel methods and compositions for the efficient expression oftransgenes in plants. In particular, the current invention provides theRS81 promoter. The RS81 promoter described herein represents aroot-specific promoter which may find wide utility in directing theexpression of potentially any gene which one desires to have expressedin a plant. This promoter represents a significant advance in that it iscapable of directing high-level expression of transgenes in plants.

The RS81 promoter sequence of the invention is exemplified by thenucleic acid sequence given in SEQ ID NO:1. However, in addition to theunmodified RS81 promoter sequence of SEQ ID NO:1, the current inventionincludes derivatives of this sequence and compositions made therefrom.In particular, the present disclosure provides the teaching for one ofskill in the art to make and use derivatives of the RS81 promoter. Forexample, the disclosure provides the teaching for one of skill in theart to delimit the functional elements within the RS81 promoter and todelete any non-essential elements. Functional elements also could bemodified to increase the utility of the sequences of the invention forany particular application. For example, a functional region within theRS81 promoter of the invention could be modified to cause or increasetissue-specific expression. Such changes could be made by site-specificmutagenesis, techniques, for example, as described below.

One important application of the RS81 promoter will be in theconstruction of vectors designed for introduction into plants by genetictransformation. By including an actin 1 intron or actin 2 intron withsuch transformation constructs comprising an RS81 promoter, one mayincrease the level of expression of coding regions operably linked tothe RS81 promoter. It is also believed that benefit will be obtained byincluding a transcriptional terminator with transgenes operably linkedto the RS81 promoter. More particularly, benefit may be realized byincluding a terminator from a gene encoding the small subunit of aribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco), and morespecifically, from a rice Rubisco gene.

I. Derivatives of the Sequences of the Invention

An important aspect of the invention provides derivatives of the maizeRS81 promoter. In particular, the current invention includes sequenceswhich have been derived from the maize RS81 promoter disclosed herein.One efficient means for preparing such derivatives comprises introducingmutations into the sequences of the invention, for example, the sequencegiven in SEQ ID NO:1. Such mutants may potentially have enhanced oraltered function relative to the native sequence or alternatively, maybe silent with regard to function.

Mutagenesis may be carried out at random and the mutagenized sequencesscreened for function in a trial-by-error procedure. Alternatively,particular sequences which provide the RS81 promoter with desirableexpression characteristics could be identified and these or similarsequences introduced into other related or non-related sequences viamutation. Similarly, non-essential elements may be deleted withoutsignificantly altering the function of the elements. It further iscontemplated that one could mutagenize these sequences in order toenhance their utility in expressing transgenes in a particular species,for example, in maize.

The means for mutagenizing a DNA segment encoding an RS81 promotersequence of the current invention are well-known to those of skill inthe art. Mutagenesis may be performed in accordance with any of thetechniques known in the art, such as, and not limited to, synthesizingan oligonucleotide having one or more mutations within the sequence of aparticular regulatory region. In particular, site-specific mutagenesisis a technique useful in the preparation of promoter mutants, throughspecific mutagenesis of the underlying DNA. The technique furtherprovides a ready ability to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 toabout 75 nucleotides or more in length is preferred, with about 10 toabout 25 or more residues on both sides of the junction of the sequencebeing altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Double stranded plasmids also areroutinely employed in site directed mutagenesis which eliminates thestep of transferring the gene of interest from a plasmid to a phage.

Site-directed mutagenesis in accordance herewith typically is performedby first obtaining a single-stranded vector or melting apart of twostrands of a double stranded vector which includes within its sequence aDNA sequence which encodes the maize RS81 promoter. An oligonucleotideprimer bearing the desired mutated sequence is prepared, generallysynthetically. This primer is then annealed with the single-strandedvector, and subjected to DNA polymerizing enzymes such as the E. colipolymerase I Klenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation. This heteroduplex vector is then used totransform or transfect appropriate cells, such as E. coli cells, andcells are selected which include recombinant vectors bearing the mutatedsequence arrangement. Vector DNA can then be isolated from these cellsand used for plant transformation. A genetic selection scheme wasdevised by Kunkel et al. (1987) to enrich for clones incorporatingmutagenic oligonucleotides. Alternatively, the use of PCR™ withcommercially available thermostable enzymes such as Taq polymerase maybe used to incorporate a mutagenic oligonucleotide primer into anamplified DNA fragment that can then be cloned into an appropriatecloning or expression vector. The PCR™-mediated mutagenesis proceduresof Tomic et al. (1990) and Upender et al. (1995) provide two examples ofsuch protocols. A PCR™ employing a thermostable ligase in addition to athermostable polymerase also may be used to incorporate a phosphorylatedmutagenic oligonucleotide into an amplified DNA fragment that may thenbe cloned into an appropriate cloning or expression vector.

The preparation of sequence variants of the selected promoter orintron-encoding DNA segments using site-directed mutagenesis is providedas a means of producing potentially useful species and is not meant tobe limiting as there are other ways in which sequence variants of DNAsequences may be obtained. For example, recombinant vectors encoding thedesired promoter sequence may be treated with mutagenic agents, such ashydroxylamine, to obtain sequence variants.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” also is intended to refer to a process thatinvolves the template-dependent extension of a primer molecule. The termtemplate-dependent process refers to nucleic acid synthesis of an RNA ora DNA molecule wherein the sequence of the newly synthesized strand ofnucleic acid is dictated by the well-known rules of complementary basepairing (see, for example, Watson and Ramstad, 1987). Typically, vectormediated methodologies involve the introduction of the nucleic acidfragment into a DNA or RNA vector, the clonal amplification of thevector, and the recovery of the amplified nucleic acid fragment.Examples of such methodologies are provided by U.S. Pat. No. 4,237,224,specifically incorporated herein by reference in its entirety. A numberof template dependent processes are available to amplify the targetsequences of interest present in a sample, such methods being well knownin the art and specifically disclosed herein below.

One efficient, targeted means for preparing mutagenized promoters orenhancers relies upon the identification of putative regulatory elementswithin the target sequence. This can be initiated by comparison with,for example, promoter sequences known to be expressed in a similarmanner. Sequences which are shared among elements with similar functionsor expression patterns are likely candidates for the binding oftranscription factors and are thus likely elements which conferexpression patterns. Confirmation of these putative regulatory elementscan be achieved by deletion analysis of each putative regulatory regionfollowed by functional analysis of each deletion construct by assay of areporter gene which is functionally attached to each construct. As such,once a starting promoter or intron sequence is provided, any of a numberof different functional deletion mutants of the starting sequence couldbe readily prepared.

As indicated above, deletion mutants of the RS81 promoter also could berandomly prepared and then assayed. With this strategy, a series ofconstructs are prepared, each containing a different portion of theclone (a subclone), and these constructs are then screened for activity.A suitable means for screening for activity is to attach a deletedpromoter construct to a selectable or screenable marker, and to isolateonly those cells expressing the marker protein. In this way, a number ofdifferent, deleted promoter constructs are identified which still retainthe desired, or even enhanced, activity. The smallest segment which isrequired for activity is thereby identified through comparison of theselected constructs. This segment may then be used for the constructionof vectors for the expression of exogenous protein.

II. Plant Transformation Constructs

The construction of vectors which may be employed in conjunction withplant transformation techniques according to the invention will be knownto those of skill of the art in light of the present disclosure (see forexample, Sambrook et al., 1989; Gelvin et al., 1990). The techniques ofthe current invention are thus not limited to any particular DNAsequences in conjunction with the RS81 promoter of the invention. Forexample, the RS81 promoter alone could be transformed into a plant withthe goal of enhancing or altering the expression of one or more genes inthe host genome.

One important use of the sequences of the invention will be in directingthe expression of a selected coding region which encodes a particularprotein or polypeptide product. However, the selected coding regionsalso may be non-expressible DNA segments, e.g., transposons such as Dsthat do not direct their own transposition. The inventors alsocontemplate that, where both an expressible gene that is not necessarilya marker gene is employed in combination with a marker gene, one mayemploy the separate genes on either the same or different DNA segmentsfor transformation. In the latter case, the different vectors aredelivered concurrently to recipient cells to maximize cotransformation.

The choice of the particular selected coding regions used in accordancewith the RS81 promoter for transformation of recipient cells will oftendepend on the purpose of the transformation. One of the major purposesof transformation of crop plants is to add commercially desirable,agronomically important traits to the plant. Such traits include, butare not limited to, herbicide resistance or tolerance; insect resistanceor tolerance; disease resistance or tolerance (viral, bacterial, fungal,nematode); stress tolerance and/or resistance, as exemplified byresistance or tolerance to drought, heat, chilling, freezing, excessivemoisture, salt stress, or oxidative stress; increased yields; foodcontent and makeup; physical appearance, male sterility; drydown;standability; prolificacy; starch properties; oil quantity and quality,and the like.

In certain embodiments, the present inventors contemplate thetransformation of a recipient cell with more than transformationconstruct. Two or more transgenes can be created in a singletransformation event using either distinct selected-protein encodingvectors, or using a single vector incorporating two or more gene codingsequences. Of course, any two or more transgenes of any description,such as those conferring, for example, herbicide, insect, disease(viral, bacterial, fungal, nematode) or drought resistance, malesterility, drydown, standability, prolificacy, starch properties, oilquantity and quality, or those increasing yield or nutritional qualitymay be employed as desired.

In other embodiments of the invention, it is contemplated that one maywish to employ replication-competent viral vectors for planttransformation. Such vectors include, for example, wheat dwarf virus(WDV) “shuttle” vectors, such as pW1-11 and PW1-GUS (Ugaki et al.,1991). These vectors are capable of autonomous replication in maizecells as well as E. coli, and as such may provide increased sensitivityfor detecting DNA delivered to transgenic cells. A replicating vectoralso may be useful for delivery of genes flanked by DNA sequences fromtransposable elements such as Ac, Ds, or Mu. It has been proposed thattransposition of these elements within the maize genome requires DNAreplication (Laufs et al., 1990). It also is contemplated thattransposable elements would be useful for introducing DNA fragmentslacking elements necessary for selection and maintenance of the plasmidvector in bacteria, e.g., antibiotic resistance genes and origins of DNAreplication. It also is proposed that use of a transposable element suchas Ac, Ds, or Mu would actively promote integration of the desired DNAand hence increase the frequency of stably transformed cells.

It further is contemplated that one may wish to co-transform plants orplant cells with 2 or more vectors. Co-transformation may be achievedusing a vector containing the marker and another gene or genes ofinterest. Alternatively, different vectors, e.g., plasmids, may containthe different genes of interest, and the plasmids may be concurrentlydelivered to the recipient cells. Using this method, the assumption ismade that a certain percentage of cells in which the marker has beenintroduced, also have received the other gene(s) of interest. Thus, notall cells selected by means of the marker, will express the otherproteins of interest which had been presented to the cells concurrently.

Vectors used for plant transformation may include, for example,plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterialartificial chromosomes) or any other suitable cloning system. It iscontemplated that utilization of cloning systems with large insertcapacities will allow introduction of large DNA sequences comprisingmore than one selected gene. Introduction of such sequences may befacilitated by use of bacterial or yeast artificial chromosomes (BACs orYACs, respectively), or even plant artificial chromosomes. For example,the use of BACs for Agrobacterium-mediated transformation was disclosedby Hamilton et al. (1996).

Particularly useful for transformation are expression cassettes whichhave been isolated from such vectors. DNA segments used for transformingplant cells will, of course, generally comprise the cDNA, gene or geneswhich one desires to introduced into and have expressed in the hostcells. These DNA segments can further include, in addition to an RS81promoter, structures such as promoters, enhancers, polylinkers, or evenregulatory genes as desired. The DNA segment or gene chosen for cellularintroduction will often encode a protein which will be expressed in theresultant recombinant cells resulting in a screenable or selectabletrait and/or which will impart an improved phenotype to the resultingtransgenic plant. However, this may not always be the case, and thepresent invention also encompasses transgenic plants incorporatingnon-expressed transgenes. Preferred components likely to be includedwith vectors used in the current invention are as follows.

(i) Regulatory Elements

Constructs prepared in accordance with the current invention willinclude an RS81 promoter or a derivative thereof. However, thesesequences may be used in the preparation of transformation constructswhich comprise a wide variety of other elements. One such application inaccordance with the instant invention will be the preparation oftransformation constructs comprising the RS81 promoter operably linkedto a selected coding region. By including an enhancer sequence with suchconstructs, the expression of the selected protein may be enhanced.These enhancers often are found 5′ to the start of transcription in apromoter that functions in eukaryotic cells, but can often be insertedin the forward or reverse orientation 5′ or 3′ to the coding sequence.In some instances these 5′ enhancing elements are introns. Deemed to beparticularly useful as enhancers are the 5′ introns of the rice actin 1and rice actin 2 genes. Examples of other enhancers which could be usedin accordance with the invention include elements from the CaMV 35Spromoter, octopine synthase genes (Ellis et al., 1987), the maizealcohol dehydrogenase gene, the maize shrunken 1 gene and promoters fromnon-plant eukaryotes (e.g., yeast; Ma et al., 1988).

Where an enhancer is used in conjunction with an RS81 promoter for theexpression of a selected protein, it is believed that it will bepreferred to place the enhancer between the promoter and the start codonof the selected coding region. However, one also could use a differentarrangement of the enhancer relative to other sequences and stillrealize the beneficial properties conferred by the enhancer. Forexample, the enhancer could be placed 5′ of the promoter region, withinthe promoter region, within the coding sequence (including within anyother intron sequences which may be present), or 3′ of the codingregion.

In addition to introns with enhancing activity, other types of elementscan influence gene expression. For example, untranslated leadersequences have been made to predict optimum or sub-optimum sequences andgenerate “consensus” and preferred leader sequences (Joshi, 1987).Preferred leader sequences are contemplated to include those which havesequences predicted to direct optimum expression of the attached codingregion, i.e., to include a preferred consensus leader sequence which mayincrease or maintain mRNA stability and prevent inappropriate initiationof translation. The choice of such sequences will be known to those ofskill in the art in light of the present disclosure. Sequences that arederived from genes that are highly expressed in plants, and in maize inparticular, will be most preferred.

Specifically contemplated for use in accordance with the presentinvention are vectors which include the ocs enhancer element. Thiselement was first identified as a 16 bp palindromic enhancer from theoctopine synthase (ocs) gene of Agrobacterium (Ellis et al., 1987), andis present in at least 10 other promoters (Bouchez et al., 1989). It isproposed that the use of an enhancer element, such as the ocs elementand particularly multiple copies of the element, may be used to increasethe level of transcription from adjacent promoters when applied in thecontext of monocot transformation.

Ultimately, the most desirable DNA segments for introduction into aplant genome may be homologous genes or gene families which encode adesired trait, and which are introduced under the control of the maizeRS81 promoter. The tissue-specific expression profile of the RS81promoter will be of particular benefit in the expression of transgenesin plants. For example, it is envisioned that a particular use of thepresent invention may be the production of transformants comprising atransgene which is expressed in a tissue-specific manner, whereby theexpression is enhanced by an actin 1 or actin 2 intron. For example,insect resistant protein may be expressed specifically in the rootswhich are targets for a number of pests including nematodes and the cornroot worm.

It also is contemplated that expression of one or more transgenes may beobtained in all tissues but roots by introducing a constitutivelyexpressed gene (all tissues) in combination with an antisense gene thatis expressed only by the RS81 promoter. Therefore, expression of anantisense transcript encoded by the constitutive promoter would preventaccumulation of the respective protein encoded by the sense transcript.Similarly, antisense technology could be used to achievetemporally-specific or inducible expression of a transgene encoded by anRS81 promoter.

It also is contemplated that it may be useful to target DNA within acell. For example, it may be useful to target introduced DNA to thenucleus as this may increase the frequency of transformation. Within thenucleus itself, it would be useful to target a gene in order to achievesite specific integration. For example, it would be useful to have agene introduced through transformation replace an existing gene in thecell.

(ii) Terminators

Transformation constructs prepared in accordance with the invention willtypically include a 3′ end DNA sequence that acts as a signal toterminate transcription and allow for the poly-adenylation of the mRNAproduced by coding sequences operably linked to the maize RS81 promoter.Terminators which are deemed to be particularly usefull in conjunctionwith the RS81 promoter are those from a gene encoding the small subunitof a ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcS), and morespecifically, from a rice rbcS gene. An exemplary rbcS terminator foruse with the maize RS81 promoter comprises the nucleic acid sequence ofSEQ ID NO:2.

Where a 3′ end other than an rbcS terminator is used in accordance withthe invention, the most preferred 3′ ends are contemplated to be thosefrom the nopaline synthase gene of Agrobacterium tumefaciens (nos 3′end) (Bevan et al., 1983), the terminator for the T7 transcript from theoctopine synthase gene of Agrobacterium tumefaciens, and the 3′ end ofthe protease inhibitor I or II genes from potato or tomato. Regulatoryelements such as Adh intron (Callis et al., 1987), sucrose synthaseintron (Vasil et al., 1989) or TMV omega element (Gallie, et al., 1989),may further be included where desired. Alternatively, one also could usea gamma coixin, oleosin 3 or other terminator from the genus Coix.

(iii) Transit or Signal Peptides

Sequences that are joined to the coding sequence of an expressed gene,which are removed post-translationally from the initial translationproduct and which facilitate the transport of the protein into orthrough intracellular or extracellular membranes, are termed transit(usually into vacuoles, vesicles, plastids and other intracellularorganelles) and signal sequences (usually to the endoplasmic reticulum,golgi apparatus and outside of the cellular membrane). By facilitatingthe transport of the protein into compartments inside and outside thecell, these sequences may increase the accumulation of gene productprotecting them from proteolytic degradation. These sequences also allowfor additional mRNA sequences from highly expressed genes to be attachedto the coding sequence of the genes. Since mRNA being translated byribosomes is more stable than naked mRNA, the presence of translatablemRNA in front of the gene may increase the overall stability of the mRNAtranscript from the gene and thereby increase synthesis of the geneproduct. Since transit and signal sequences are usuallypost-translationally removed from the initial translation product, theuse of these sequences allows for the addition of extra translatedsequences that may not appear on the final polypeptide. It further iscontemplated that targeting of certain proteins may be desirable inorder to enhance the stability of the protein (U.S. Pat. No. 5,545,818,incorporated herein by reference in its entirety).

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This generally will be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit, or signal, peptide willtransport the protein to a particular intracellular, or extracellulardestination, respectively, and will then be post-translationallyremoved.

A particular example of such a use concerns the direction of a proteinconferring herbicide resistance, such as a mutant EPSPS protein, to aparticular organelle such as the chloroplast rather than to thecytoplasm. This is exemplified by the use of the rbcS transit peptide,the chloroplast transit peptide described in U.S. Pat. No. 5,728,925, orthe optimized transit peptide described in U.S. Pat. No. 5,510,471,which confers plastid-specific targeting of proteins. In addition, itmay be desirable to target certain genes responsible for male sterilityto the mitochondria, or to target certain genes for resistance tophytopathogenic organisms to the extracellular spaces, or to targetproteins to the vacuole. A further use concerns the direction of enzymesinvolved in amino acid biosynthesis or oil synthesis to the plastid.Such enzymes include dihydrodipicolinic acid synthase which maycontribute to increasing lysine content of a feed.

(iv) Marker Genes

One application of the maize RS81 promoter of the current invention willbe in the expression of marker proteins. By employing a selectable orscreenable marker gene as, or in addition to, the gene of interest, onecan provide or enhance the ability to identify transformants. “Markergenes” are genes that impart a distinct phenotype to cells expressingthe marker gene and thus allow such transformed cells to bedistinguished from cells that do not have the marker. Such genes mayencode either a selectable or screenable marker, depending on whetherthe marker confers a trait which one can “select” for by chemical means,i.e., through the use of a selective agent (e.g., a herbicide,antibiotic, or the like), or whether it is simply a trait that one canidentify through observation or testing, i.e., by “screening” (e.g., thegreen fluorescent protein). Of course, many examples of suitable markergenes are known to the art and can be employed in the practice of theinvention.

Included within the terms selectable or screenable marker genes also aregenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude marker genes which encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes which canbe detected by their catalytic activity. Secretable proteins fall into anumber of classes, including small, diffusible proteins detectable,e.g., by ELISA; small active enzymes detectable in extracellularsolution (e.g., α-amylase, β-lactamase, phosphinothricinacetyltransferase); and proteins that are inserted or trapped in thecell wall (e.g., proteins that include a leader sequence such as thatfound in the expression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a protein that becomes sequestered in the cell wall, and whichprotein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

One example of a protein suitable for modification in this manner isextensin, or hydroxyproline rich glycoprotein (HPRG). The use of maizeHPRG (Steifel et al., 1990) is preferred, as this molecule is wellcharacterized in terms of molecular biology, expression and proteinstructure. However, any one of a variety of extensins and/orglycine-rich wall proteins (Keller et al., 1989) could be modified bythe addition of an antigenic site to create a screenable marker.

One exemplary embodiment of a secretable screenable marker concerns theuse of a maize sequence encoding the wall protein HPRG, modified toinclude a 15 residue epitope from the pro-region of murineinterleukin-1-β(IL-1-β). However, virtually any detectable epitope maybe employed in such embodiments, as selected from the extremely widevariety of antigen:antibody combinations known to those of skill in theart. The unique extracellular epitope, whether derived from IL-1β or anyother protein or epitopic substance, can then be straightforwardlydetected using antibody labeling in conjunction with chromogenic orfluorescent adjuncts.

1. Selectable Markers

Many selectable marker coding regions may be used in connection with theRS81 promoter of the present invention including, but not limited to,neo (Potrykus et al., 1985) which provides kanamycin resistance and canbe selected for using kanamycin, G418, paromomycin, etc.; bar, whichconfers bialaphos or phosphinothricin resistance; a mutant EPSP synthaseprotein (Hinchee et al., 1988) conferring glyphosate resistance; anitrilase such as bxn from Klebsiella ozaenae which confers resistanceto bromoxynil (Stalker et al., 1988); a mutant acetolactate synthase(ALS) which confers resistance to imidazolinone, sulfonylurea or otherALS inhibiting chemicals (European Patent Application 154,204, 1985); amethotrexate resistant DHFR (Thillet et al., 1988), a dalapondehalogenase that confers resistance to the herbicide dalapon; or amutated anthranilate synthase that confers resistance to 5-methyltryptophan. Where a mutant EPSP synthase is employed, additional benefitmay be realized through the incorporation of a suitable chloroplasttransit peptide, CTP (U.S. Pat. No. 5,188,642) or OTP (U.S. Pat. No.5,633,448) and use of a modified maize EPSPS (PCT Application WO97/04103).

An illustrative embodiment of selectable markers capable of being usedin systems to select transformants are the enzyme phosphinothricinacetyltransferase, such as bar from Streptomyces hygroscopicus or patfrom Streptomyces viridochromogenes. The enzyme phosphinothricin acetyltransferase (PAT) inactivates the active ingredient in the herbicidebialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase,(Murakami et al., 1986; Twell et al., 1989) causing rapid accumulationof ammonia and cell death.

Where one desires to employ bialaphos resistance in the practice of theinvention, the inventor has discovered that particularly useful genesfor this purpose are the bar or pat genes obtainable from species ofStreptomyces (e.g., ATCC No. 21,705). The cloning of the bar gene hasbeen described (Murakami et al., 1986; Thompson et al., 1987) as has theuse of the bar gene in the context of plants (De Block et al., 1987; DeBlock et al., 1989; U.S. Pat. No. 5,550,318).

2. Screenable Markers

Screenable markers that may be employed include a β-glucuronidase (GUS)or uidA gene which encodes an enzyme for which various chromogenicsubstrates are known; an R-locus gene, which encodes a product thatregulates the production of anthocyanin pigments (red color) in planttissues (Dellaporta et al., 1988); a β-lactamase gene (Sutcliffe, 1978),which encodes an enzyme for which various chromogenic substrates areknown (e.g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowskyet al., 1983) which encodes a catechol dioxygenase that can convertchromogenic catechols; an α-amylase gene (Ikuta et al., 1990); atyrosinase gene (Katz et al., 1983) which encodes an enzyme capable ofoxidizing tyrosine to DOPA and dopaquinone which in turn condenses toform the easily-detectable compound melanin; a β-galactosidase gene,which encodes an enzyme for which there are chromogenic substrates; aluciferase (lux) gene (Ow et al., 1986), which allows forbioluminescence detection; an aequorin gene (Prasher et al., 1985) whichmay be employed in calcium-sensitive bioluminescence detection; or agene encoding for green fluorescent protein (Sheen et al., 1995;Haseloff et al., 1997; Reichel et al, 1996; Tian et al., 1997; WO97/41228).

Genes from the maize R gene complex are contemplated to be particularlyuseful as screenable markers. The R gene complex in maize encodes aprotein that acts to regulate the production of anthocyanin pigments inmost seed and plant tissue. Maize strains can have one, or as many asfour, R alleles which combine to regulate pigmentation in adevelopmental and tissue specific manner. Thus, an R gene introducedinto such cells will cause the expression of a red pigment and, ifstably incorporated, can be visually scored as a red sector. If a maizeline carries dominant alleles for genes encoding for the enzymaticintermediates in the anthocyanin biosynthetic pathway (C2, A1, A2, Bz1and Bz2), but carries a recessive allele at the R locus, transformationof any cell from that line with R will result in red pigment formation.Exemplary lines include Wisconsin 22 which contains the rg-Stadlerallele and TR112, a K55 derivative which is r-g, b, P1. Alternatively,any genotype of maize can be utilized if the C1 and R alleles areintroduced together.

It further is proposed that R gene regulatory regions may be employed inchimeric constructs in order to provide mechanisms for controlling theexpression of chimeric genes. More diversity of phenotypic expression isknown at the R locus than at any other locus (Coe et al., 1988). It iscontemplated that regulatory regions obtained from regions 5′ to thestructural R gene would be valuable in directing the expression of genesfor, e.g., insect resistance, herbicide tolerance or other proteincoding regions. For the purposes of the present invention, it isbelieved that any of the various R gene family members may besuccessfully employed (e.g., P, S, Lc, etc.). However, the mostpreferred will generally be Sn (particularly Sn:bol3). Sn is a dominantmember of the R gene complex and is functionally similar to the R and Bloci in that Sn controls the tissue specific deposition of anthocyaninpigments in certain seedling and plant cells, therefore, its phenotypeis similar to R.

Another screenable marker contemplated for use in the present inventionis firefly luciferase, encoded by the lux gene. The presence of the luxgene in transformed cells may be detected using, for example, X-rayfilm, scintillation counting, fluorescent spectrophotometry, low-lightvideo cameras, photon counting cameras or multiwell luminometry. It alsois envisioned that this system may be developed for populationalscreening for bioluminescence, such as on tissue culture plates, or evenfor whole plant screening. The gene which encodes green fluorescentprotein (GFP) is contemplated as a particularly useful reporter gene(Sheen et al., 1995; Haseloff et al., 1997; Reichel et al., 1996; Tianet al., 1997; WO 97/41228). Expression of green fluorescent protein maybe visualized in a cell or plant as fluorescence following illuminationby particular wavelengths of light. Where use of a screenable markergene such as lux or GFP is desired, the inventors contemplated thatbenefit may be realized by creating a gene fusion between the screenablemarker gene and a selectable marker gene, for example, a GFP-NPTII genefusion. This could allow, for example, selection of transformed cellsfollowed by screening of transgenic plants or seeds.

III. Exogenous Genes for Modification of Plant Phenotypes

A particularly important advance of the present invention is that itprovides methods and compositions for the efficient expression ofselected proteins in plant cells. In particular, the current inventionprovides an RS81 promoter for the expression of selected proteins inplants. By including an enhancer with transformation constructscomprising the RS81 promoter, increased expression of selected proteinscan be realized following introduction of the transformation constructinto a host plant cell.

The choice of a selected protein for expression in a plant host cell inaccordance with the invention will depend on the purpose of thetransformation. One of the major purposes of transformation of cropplants is to add commercially desirable, agronomically important traitsto the plant. Such traits include, but are not limited to, herbicideresistance or tolerance; insect resistance or tolerance; diseaseresistance or tolerance (viral, bacterial, fungal, nematode); stresstolerance and/or resistance, as exemplified by resistance or toleranceto drought, heat, chilling, freezing, excessive moisture, salt stressand oxidative stress; increased yields; food content and makeup;physical appearance; male sterility; drydown; standability; prolificacy;starch quantity and quality; oil quantity and quality; protein qualityand quantity; amino acid composition; and the like.

In certain embodiments of the invention, transformation of a recipientcell may be carried out with more than one exogenous (selected) gene. Asused herein, an “exogenous coding region” or “selected coding region” isa coding region not normally found in the host genome in an identicalcontext. By this, it is meant that the coding region may be isolatedfrom a different species than that of the host genome, or alternatively,isolated from the host genome, but is operably linked to one or moreregulatory regions which differ from those found in the unaltered,native gene. Two or more exogenous coding regions also can be suppliedin a single transformation event using either distincttransgene-encoding vectors, or using a single vector incorporating twoor more coding sequences. For example, plasmids bearing the bar and aroAexpression units in either convergent, divergent, or colinearorientation, are considered to be particularly useful. Further preferredcombinations are those of an insect resistance gene, such as a Bt gene,along with a protease inhibitor gene such as pinII, or the use of bar incombination with either of the above genes. Of course, any two or moretransgenes of any description, such as those conferring herbicide,insect, disease (viral, bacterial, fungal, nematode) or droughtresistance, male sterility, drydown, standability, prolificacy, starchproperties, oil quantity and quality, or those increasing yield ornutritional quality may be employed as desired.

(i) Herbicide Resistance

The DNA segments encoding phosphinothricin acetyltransferase (bar andpat), EPSP synthase encoding genes conferring resistance to glyphosate,the glyphosate degradative enzyme gene gox encoding glyphosateoxidoreductase, deh (encoding a dehalogenase enzyme that inactivatesdalapon), herbicide resistant (e.g., sulfonylurea and imidazolinone)acetolactate synthase, and bxn genes (encoding a nitrilase enzyme thatdegrades bromoxynil) are examples of herbicide resistant genes for usein transformation. The bar and pat genes code for an enzyme,phosphinothricin acetyltransferase (PAT), which inactivates theherbicide phosphinothricin and prevents this compound from inhibitingglutamine synthetase enzymes. The enzyme 5-enolpyruvylshikimate3-phosphate synthase (EPSP Synthase), is normally inhibited by theherbicide N-(phosphonomethyl)glycine (glyphosate). However, genes areknown that encode glyphosate-resistant EPSP synthase enzymes. Thesegenes are particularly contemplated for use in plant transformation. Thedeh gene encodes the enzyme dalapon dehalogenase and confers resistanceto the herbicide dalapon. The bxn gene codes for a specific nitrilaseenzyme that converts bromoxynil to a non-herbicidal degradation product.

(ii) Insect Resistance

Potential insect resistance genes that can be introduced includeBacillus thuringiensis crystal toxin genes or Bt genes (Watrud et al.,1985). Bt genes may provide resistance to lepidopteran or coleopteranpests such as European Corn Borer (ECB). Preferred Bt or other types ofinsect resistance genes for use with the current invention will haveactivity against pests capable of infesting the root systems of cropplants, for example, the northern corn rootworm (Diabrotica barberi),southern corn rootworm (Diabrotica undecimpunctata), Western cornrootworm (Diabrotica virgifera), black cutworm (Agrotis ipsilon), glassycutworm (Crymodes devastator), dingy cutworm (Feltia ducens), claybackedcutworm (Agrotis gladiaria), wireworm (Melanotus spp., Aeolusmellillus), wheat wireworm (Aeolus mancus), sand wireworm (Horistonotusuhlerii), maize billbug (Sphenophorus maidis), timothy billbug(Sphenophorus zeae), bluegrass billbug (Sphenophorus parvulus), southerncorn billbug (Sphenophorus callosus), white grubs (Phyllogphaga spp.),corn root aphid (Anuraphis maidiradicis), seedcorn maggot (Deliaplatura), grape colaspis (Colaspis brunnea), seedcorn beetle(Stenolophus lecontei), and slender seedcorn beetle (Cliviniaimpressifrons).

It is contemplated that preferred Bt genes for use in the transformationprotocols disclosed herein will be those in which the coding sequencehas been modified to effect increased expression in plants, and moreparticularly, in maize. Means for preparing synthetic genes are wellknown in the art and are disclosed in, for example, U.S. Pat. No.5,500,365 and U.S. Pat. Number No. 5,689,052, each of the disclosures ofwhich are specifically incorporated herein by reference in theirentirety. Examples of such modified Bt toxin genes include a syntheticBt CryIA(b) gene (Perlak et al., 1991), and the synthetic CryIA(c) genetermed 1800b (PCT Application WO 95/06128). Some examples of other Bttoxin genes known to those of skill in the art are given in Table 1below.

TABLE 1 Bacillus thuringiensis δ-Endotoxin Genes^(a) New NomenclatureOld Nomenclature GenBank Accession Cry1Aa CryIA(a) M11250 Cry1AbCryIA(b) M13898 Cry1Ac CryIA(c) M11068 Cry1Ad CryIA(d) M73250 Cry1AeCryIA(e) M65252 Cry1Ba CryIB X06711 Cry1Bb ET5 L32020 Cry1Bc PEG5 Z46442Cry1Bd CryE1 U70726 Cry1Ca CryIC X07518 Cry1Cb CryIC(b) M97880 Cry1DaCryID X54160 Cry1Db PrtB Z22511 Cry1Ea CryIE X53985 Cry1Eb CryIE(b)M73253 Cry1Fa CryIF M63897 Cry1Fb PrtD Z22512 Cry1Ga PrtA Z22510 Cry1GbCryH2 U70725 Cry1Na PrtC Z22513 Cry1Hb U35780 Cry1Ia CryV X62821 Cry1IbCryV U07642 Cry1Ja ET4 L32019 Cry1Jb ET1 U31527 Cry1K U28801 Cry2AaCryIIA M31738 Cry2Ab CryIIB M23724 Cry2Ac CryIIC X57252 Cry3A CryIIIAM22472 Cry3Ba CryIIIB X17123 Cry3Bb CryIIIB2 M89794 Cry3C CryIIID X59797Cry4A CryIVA Y00423 Cry4B CryIVB X07423 Cry5Aa CryVA(a) L07025 Cry5AbCryVA(b) L07026 Cry6A CryVIA L07022 Cry6B CryVIB L07024 Cry7Aa CryIIICM64478 Cry7Ab CryIIICb U04367 Cry8A CryIIIE U04364 Cry8B CryIIIG U04365Cry8C CryIIIF U04366 Cry9A CryIG X58120 Cry9B CryIX X75019 Cry9C CryIHZ37527 Cry10A CryIVC M12662 Cry11A CryIVD M31737 Cry11B Jeg80 X86902Cry12A CryVB L07027 Cry13A CryVC L07023 Cry14A CryVD U13955 Cry15A 34kDaM76442 Cry16A cbm71 X94146 Cry17A cbm71 X99478 Cry18A CryBP1 X99049Cry19A Jeg65 Y08920 Cyt1Aa CytA X03182 Cyt1Ab CytM X98793 Cyt2A CytBZ14147 Cyt2B CytB U52043 ^(a)Adapted from:http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html

Protease inhibitors also may provide insect resistance (Johnson et al.,1989), and thus will have utility in plant transformation. The use of aprotease inhibitor II gene, pinII, from tomato or potato is envisionedto be particularly useful. Even more advantageous is the use of a pinIIgene in combination with a Bt toxin gene, the combined effect of whichhas been discovered to produce synergistic insecticidal activity. Othergenes which encode inhibitors of the insect's digestive system, or thosethat encode enzymes or co-factors that facilitate the production ofinhibitors, also may be useful. This group may be exemplified byoryzacystatin and amylase inhibitors such as those from wheat andbarley.

Also, genes encoding lectins may confer additional or alternativeinsecticide properties. Lectins (originally termed phytohemagglutinins)are multivalent carbohydrate-binding proteins which have the ability toagglutinate red blood cells from a range of species. Lectins have beenidentified recently as insecticidal agents with activity againstweevils, ECB and rootworm (Murdock et al., 1990; Czapla & Lang, 1990).Lectin genes contemplated to be useful include, for example, barley andwheat germ agglutinin (WGA) and rice lectins (Gatehouse et al., 1984),with WGA being preferred.

Genes controlling the production of large or small polypeptides activeagainst insects when introduced into the insect pests, such as, e.g.,lytic peptides, peptide hormones and toxins and venoms, form anotheraspect of the invention. For example, it is contemplated that theexpression of juvenile hormone esterase, directed towards specificinsect pests, also may result in insecticidal activity, or perhaps causecessation of metamorphosis (Hammock et al., 1990).

Transgenic plants expressing genes which encode enzymes that affect theintegrity of the insect cuticle form yet another aspect of theinvention. Such genes include those encoding, e.g., chitinase,proteases, lipases and also genes for the production of nikkomycin, acompound that inhibits chitin synthesis, the introduction of any ofwhich is contemplated to produce insect resistant plants. Genes thatcode for activities that affect insect molting, such as those affectingthe production of ecdysteroid UDP-glucosyl transferase, also fall withinthe scope of the useful transgenes of the present invention.

Genes that code for enzymes that facilitate the production of compoundsthat reduce the nutritional quality of the host plant to insect pestsalso are encompassed by the present invention. It may be possible, forinstance, to confer insecticidal activity on a plant by altering itssterol composition. Sterols are obtained by insects from their diet andare used for hormone synthesis and membrane stability. Therefore,alterations in plant sterol composition by expression of novel genes,e.g., those that directly promote the production of undesirable sterolsor those that convert desirable sterols into undesirable forms, couldhave a negative effect on insect growth and/or development and henceendow the plant with insecticidal activity. Lipoxygenases are naturallyoccurring plant enzymes that have been shown to exhibit anti-nutritionaleffects on insects and to reduce the nutritional quality of their diet.Therefore, further embodiments of the invention concern transgenicplants with enhanced lipoxygenase activity which may be resistant toinsect feeding.

Tripsacum dactyloides is a species of grass that is resistant to certaininsects, including corn root worm. It is anticipated that genes encodingproteins that are toxic to insects or are involved in the biosynthesisof compounds toxic to insects will be isolated from Tripsacum and thatthese novel genes will be useful in conferring resistance to insects. Itis known that the basis of insect resistance in Tripsacum is genetic,because said resistance has been transferred to Zea mays via sexualcrosses (Branson and Guss, 1972). It further is anticipated that othercereal, monocot or dicot plant species may have genes encoding proteinsthat are toxic to insects which would be useful for producing insectresistant corn plants.

Further genes encoding proteins characterized as having potentialinsecticidal activity also may be used as transgenes in accordanceherewith. Such genes include, for example, the cowpea trypsin inhibitor(CpTI; Hilder et al., 1987) which may be used as a rootworm deterrent;genes encoding avermectin (Avermectin and Abamectin., Campbell, W. C.,Ed., 1989; Ikeda et al., 1987) which may prove particularly useful as acorn rootworm deterrent; ribosome inactivating protein genes; and evengenes that regulate plant structures. Transgenic maize includinganti-insect antibody genes and genes that code for enzymes that canconvert a non-toxic insecticide (pro-insecticide) applied to the outsideof the plant into an insecticide inside the plant also are contemplated.

(iii) Environment or Stress Resistance

Improvement of a plants ability to tolerate various environmentalstresses such as, but not limited to, drought, excess moisture,chilling, freezing, high temperature, salt, and oxidative stress, alsocan be effected through expression of novel genes. It is proposed thatbenefits may be realized in terms of increased resistance to freezingtemperatures through the introduction of an “antifreeze” protein such asthat of the Winter Flounder (Cutler et al, 1989) or synthetic genederivatives thereof. Improved chilling tolerance also may be conferredthrough increased expression of glycerol-3-phosphate acetyltransferasein chloroplasts (Wolter et al., 1992). Resistance to oxidative stress(often exacerbated by conditions such as chilling temperatures incombination with high light intensities) can be conferred by expressionof superoxide dismutase (Gupta et al., 1993), and may be improved byglutathione reductase (Bowler et al., 1992). Such strategies may allowfor tolerance to freezing in newly emerged fields as well as extendinglater maturity higher yielding varieties to earlier relative maturityzones.

It is contemplated that the expression of novel genes that favorablyeffect plant water content, total water potential, osmotic potential,and turgor will enhance the ability of the plant to tolerate drought. Asused herein, the terms “drought resistance” and “drought tolerance” areused to refer to a plants increased resistance or tolerance to stressinduced by a reduction in water availability, as compared to normalcircumstances, and the ability of the plant to function and survive inlower-water environments. In this aspect of the invention it isproposed, for example, that the expression of genes encoding for thebiosynthesis of osmotically-active solutes, such as polyol compounds,may impart protection against drought. Within this class are genesencoding for mannitol-L-phosphate dehydrogenase (Lee and Saier, 1982)and trehalose-6-phosphate synthase (Kaasen et al., 1992). Through thesubsequent action of native phosphatases in the cell or by theintroduction and coexpression of a specific phosphatase, theseintroduced genes will result in the accumulation of either mannitol ortrehalose, respectively, both of which have been well documented asprotective compounds able to mitigate the effects of stress. Mannitolaccumulation in transgenic tobacco has been verified and preliminaryresults indicate that plants expressing high levels of this metaboliteare able to tolerate an applied osmotic stress (Tarczynski et al., 1992,1993). Altered water utilization in transgenic corn producing mannitolalso has been demonstrated (U.S. Pat. No. 5,780,709).

Similarly, the efficacy of other metabolites in protecting either enzymefunction (e.g., alanopine or propionic acid) or membrane integrity(e.g., alanopine) has been documented (Loomis et al., 1989), andtherefore expression of genes encoding for the biosynthesis of thesecompounds might confer drought resistance in a manner similar to orcomplimentary to mannitol. Other examples of naturally occurringmetabolites that are osmotically active and/or provide some directprotective effect during drought and/or desiccation include fructose,erythritol (Coxson et al., 1992), sorbitol, dulcitol (Karsten et al.,1992), glucosylglycerol (Reed et al., 1984; Erdmann et al., 1992),sucrose, stachyose (Koster and Leopold, 1988; Blackman et al., 1992),raffinose (Bernal-Lugo and Leopold, 1992), proline (Rensburg et al.,1993), glycine betaine, ononitol and pinitol (Vernon and Bohnert, 1992).Continued canopy growth and increased reproductive fitness during timesof stress will be augmented by introduction and expression of genes suchas those controlling the osmotically active compounds discussed aboveand other such compounds. Currently preferred genes which promote thesynthesis of an osmotically active polyol compound are genes whichencode the enzymes mannitol-1-phosphate dehydrogenase,trehalose-6-phosphate synthase and myoinositol 0-methyltransferase.

It is contemplated that the expression of specific proteins also mayincrease drought tolerance. Three classes of Late Embryogenic Proteinshave been assigned based on structural similarities (see Dure et al.,1989). All three classes of LEAs have been demonstrated in maturing(ie., desiccating) seeds. Within these 3 types of LEA proteins, theType-II (dehydrin-type) have generally been implicated in drought and/ordesiccation tolerance in vegetative plant parts (i.e., Mundy and Chua,1988; Piatkowski et al., 1990; Yamaguchi-Shinozaki et al., 1992).Recently, expression of a Type-III LEA (HVA-1) in tobacco was found toinfluence plant height, maturity and drought tolerance (Fitzpatrick,1993). In rice, expression of the HVA-1 gene influenced tolerance towater deficit and salinity (Xu et al., 1996). Expression of structuralgenes from all three LEA groups may therefore confer drought tolerance.Other types of proteins induced during water stress include thiolproteases, aldolases and transmembrane transporters (Guerrero et al.,1990), which may confer various protective and/or repair-type functionsduring drought stress. It also is contemplated that genes that effectlipid biosynthesis and hence membrane composition might also be usefulin conferring drought resistance on the plant.

Many of these genes for improving drought resistance have complementarymodes of action. Thus, it is envisaged that combinations of these genesmight have additive and/or synergistic effects in improving droughtresistance in crop plants such as, for example, corn. Many of thesegenes also improve freezing tolerance (or resistance); the physicalstresses incurred during freezing and drought are similar in nature andmay be mitigated in similar fashion. Benefit may be conferred viaconstitutive expression of these genes, but the preferred means ofexpressing these novel genes may be through the use of a turgor-inducedpromoter (such as the promoters for the turgor-induced genes describedin Guerrero et al., 1990 and Shagan et al., 1993 which are incorporatedherein by reference). Spatial and temporal expression patterns of thesegenes may enable plants to better withstand stress.

It is proposed that expression of genes that are involved with specificmorphological traits that allow for increased water extractions fromdrying soil would be of benefit. For example, introduction andexpression of genes that alter root characteristics may enhance wateruptake. It also is contemplated that expression of genes that enhancereproductive fitness during times of stress would be of significantvalue. For example, expression of genes that improve the synchrony ofpollen shed and receptiveness of the female flower parts, i.e., silks,would be of benefit. In addition it is proposed that expression of genesthat minimize kernel abortion during times of stress would increase theamount of grain to be harvested and hence be of value.

Given the overall role of water in determining yield, it is contemplatedthat enabling corn and other crop plants to utilize water moreefficiently, through the introduction and expression of novel genes,will improve overall performance even when soil water availability isnot limiting. By introducing genes that improve the ability of plants tomaximize water usage across a full range of stresses relating to wateravailability, yield stability or consistency of yield performance may berealized.

(iv) Disease Resistance

It is proposed that increased resistance to diseases may be realizedthrough introduction of genes into plants, for example, intomonocotyledonous plants such as maize. It is possible to produceresistance to diseases caused by viruses, bacteria, fungi and nematodes.It also is contemplated that control of mycotoxin producing organismsmay be realized through expression of introduced genes.

Resistance to viruses may be produced through expression of novel genes.For example, it has been demonstrated that expression of a viral coatprotein in a transgenic plant can impart resistance to infection of theplant by that virus and perhaps other closely related viruses (Cuozzo etal., 1988, Hemenway et al., 1988, Abel et al., 1986). It is contemplatedthat expression of antisense genes targeted at essential viral functionsalso may impart resistance to viruses. For example, an antisense genetargeted at the gene responsible for replication of viral nucleic acidmay inhibit replication and lead to resistance to the virus. It isbelieved that interference with other viral functions through the use ofantisense genes also may increase resistance to viruses. Similarly,ribozymes could be used in this context. Further, it is proposed that itmay be possible to achieve resistance to viruses through otherapproaches, including, but not limited to the use of satellite viruses.Examples of viral and viral-like diseases, for which one could introduceresistance to in a transgenic plant in accordance with the instantinvention, are listed below, in Table 2.

TABLE 2 Plant Virus and Virus-like Diseases DISEASE CAUSATIVE AGENTAmerican wheat striate American wheat striate mosaic virus mosaic (wheatstriate mosaic) (AWSMV) Barley stripe mosaic Barley stripe mosaic virus(BSMV) Barley yellow dwarf Barley yellow dwarf virus (BYDV) Brome mosaicBrome mosaic virus (BMV) Cereal chlorotic mottle* Cereal chloroticmottle virus (CCMV) Corn chlorotic vein Corn chlorotic vein bandingvirus (CCVBV) banding (Braizilian maize mosaic)¹ mosaic) Corn lethalnecrosis Virus complex (Maize chlorotic mottle virus(MCMV) and Maizedwarf mosaic virus (MDMV) A or B or Wheat streak mosaic virus(WSMV))Cucumber mosaic Cucumber mosaic virus (CMV) Cynodon chlorotic Cynodonchlorotic streak virus (CCSV) streak*^(,1) Johnsongrass mosaicJohnsongrass mosaic virus (JGMV) Maize bushy stunt Mycoplasma-likeorganism (MLO) associated Maize chlorotic dwarf Maize chlorotic dwarfvirus (MCDV) Maize chlorotic mottle Maize chlorotic mottle virus (MCMV)Maize dwarf mosaic Maize dwarf mosaic virus (MDMV) strains A, D, E and FMaize leaf fleck Maize leaf fleck virus (MLFV) Maize line* Maize linevirus (MLV) Maize mosaic (corn leaf Maize mosaic virus (MMV) stripe,enanismo rayado) Maize mottle and Maize mottle and chlorotic stuntvirus* chlorotic stunt¹ Maize pellucid ringspot* Maize pellucid ringspotvirus (MPRV) Maize raya gruesa*^(,1) Maize raya gruesa virus (MRGV)maize rayado fino* Maize rayado fino virus (MRFV) (fine stripingdisease) Maize red leaf and red Mollicute stripe* Maize red stripe*Maize red stripe virus (MRSV) Maize ring mottle* Maize ring mottle virus(MRMV) Maize rio IV* Maize rio cuarto virus (MRCV) Maize rough dwarf*Maize rough dwarf virus (MRDV) (nanismo ruvido) (= Cereal tilleringdisease virus*) Maize sterile stunt* Maize sterile stunt virus (strainsof barley yellow striate virus) Maize streak* Maize streak virus (MSV)Maize stripe (maize Maize stripe virus chlorotic stripe, maize hojablanca) Maize stunting*^(,1) Maize stunting virus Maize tassel abortion*Maize tassel abortion virus (MTAV) Maize vein enation* Maize veinenation virus (MVEV) Maize wallaby ear* Maize wallaby ear virus (MWEV)Maize white leaf* Maize white leaf virus Maize white line mosaic Maizewhite line mosaic virus (MWLMV) Millet red leaf* Millet red leaf virus(MRLV) Northern cereal mosaic* Northern cereal mosaic virus (NCMV) Oatpseudorosette* Oat pseudorosette virus (zakuklivanie) Oat sterile dwarf*Oat sterile dwarf virus (OSDV) Rice black-streaked Rice black-streakeddwarf virus (RBSDV) dwarf* Rice stripe* Rice stripe virus (RSV) Sorghummosaic Sorghum mosaic virus (SrMV), formerly sugarcane mosaic virus(SCMV) strains H, I and M Sugarcane Fiji disease* Sugarcane Fiji diseasevirus (FDV) Sugarcane mosaic Sugarcane mosaic virus (SCMV) strains A, B,D, E, SC, BC, Sabi and MB (formerly MDMV-B) Vein enation*^(,1) VirusWheat spot mosaic¹ Wheat spot mosaic virus (WSMV) *Not known to occurnaturally on corn in the United States. ¹Minor viral disease.

It is proposed that increased resistance to diseases caused by bacteriaand fungi also may be realized through introduction of novel genes. Itis contemplated that genes encoding so-called “peptide antibiotics,”pathogenesis related (PR) proteins, toxin resistance, and proteinsaffecting host-pathogen interactions such as morphologicalcharacteristics will be useful. Peptide antibiotics are polypeptidesequences which are inhibitory to growth of bacteria and othermicroorganisms. For example, the classes of peptides referred to ascecropins and magainins inhibit growth of many species of bacteria andfungi. It is proposed that expression of PR proteins in monocotyledonousplants such as maize may be useful in conferring resistance to bacterialdisease. These genes are induced following pathogen attack on a hostplant and have been divided into at least five classes of proteins (Bol,Linthorst, and Cornelissen, 1990). Included amongst the PR proteins areβ-1, 3-glucanases, chitinases, and osmotin and other proteins that arebelieved to function in plant resistance to disease organisms. Othergenes have been identified that have antifungal properties, e.g., UDA(stinging nettle lectin) and hevein (Broakaert et al., 1989;Barkai-Golan et al., 1978). It is known that certain plant diseases arecaused by the production of phytotoxins. It is proposed that resistanceto these diseases would be achieved through expression of a novel genethat encodes an enzyme capable of degrading or otherwise inactivatingthe phytotoxin. It also is contemplated that expression of novel genesthat alter the interactions between the host plant and pathogen may beuseful in reducing the ability of the disease organism to invade thetissues of the host plant, e.g., an increase in the waxiness of the leafcuticle or other morphological characteristics. Examples of bacterialand fungal diseases, including downy mildews, for which one couldintroduce resistance to in a transgenic plant in accordance with theinstant invention, are listed below, in Tables 3, 4 and 5.

TABLE 3 Plant Bacterial Diseases DISEASE CAUSATIVE AGENT Bacterial leafblight Pseudomonas avenae subsp. avenae and stalk rot Bacterial leafspot Xanthomonas campestris pv. holcicola Bacterial stalk rotEnterobacter dissolvens = Erwinia dissolvens Bacterial stalk and Erwiniacarotovora subsp. carotovora, top rot Erwinia chrysanthemi pv. zeaeBacterial stripe Pseudomonas andropogonis Chocolate spot Pseudomonassyringae pv. coronafaciens Goss's bacterial wilt and Clavibactermichiganensis subsp. nebraskensis and blight (leaf freckles =Corynebacterium michiganense pv. and wilt) nebraskense Holcus spotPseudomonas syringae pv. syringae Purple leaf sheath Hemiparasiticbacteria + (See under Fungi) Seed rot-seedling blight Bacillus subtilisStewart's disease Pantoea stewartii = Erwinia stewartii (bacterial wilt)Corn stunt Spiroplasma kunkelii (achapparramiento, maize stunt, MesaCentral or Rio Grande maize stunt)

TABLE 4 Plant Fungal Diseases DISEASE PATHOGEN Anthrocnose leaf blightColletotrichum graminicola (teleomorph: and anthracnose stalk rotGlomerella graminicola Politis), Glomerella tucumanensis (anamorph:Glomerella falcatum Went) Aspergillus ear and Aspergillus flavus Link:Fr. kernel rot Banded leaf and Rhizoctonia solani Kühn = Rhizoctoniasheath spot* microsclerotia J. Matz (telomorph: Thanatephorus cucumeris)Black bundle disease Acremonium strictum W. Gams = Cephalosporiumacremonium Auct. non Corda Black kernel rot* Lasiodiplodia theobromae =Botryodiplodia theobromae Borde blanco* Marasmiellus sp. Brown spot(black spot, Physoderma maydis stalk rot) Cephalosporium kernelAcremonium strictum = Cephalosporium rot acremonium Charcoal rotMacrophomina phaseolina Corticium ear rot* Thanatephorus cucumeris =Corticium sasakii Curvularia leaf spot Curvularia clavata, C.eragrostidis, = C. maculans (teleomorph: Cochliobolus eragrostidis),Curvularia inaequalis, C. intermedia (teleomorph: Cochliobolusintermedius), Curvularia lunata (teleomorph: Cochliobolus lunatus),Curvularia pallescens (teleomorph: Cochliobolus pallescens), Curvulariasenegalensis, C. tuberculata (teleomorph: Cochliobolus tuberculatus)Didymella leaf spot* Didymella exitalis Diplodia ear rot and Diplodiafrumenti (teleomorph: stalk rot Botryosphaeria festucae) Diplodia earrot, stalk Diplodia maydis = Stenocarpella maydis rot, seed rot andseedling blight Diplodia leaf spot or Stenocarpella macrospora =Diplodia leaf streak macrospora *Not known to occur naturally on corn inthe United States.

TABLE 5 Plant Downy Mildews DISEASE CAUSATIVE AGENT Brown stripe downySclerophthora rayssiae var. zeae mildew* Crazy top downy mildewSclerophthora macrospora = Sclerospora macrospora Green ear downy mildewSclerospora graminicola (graminicola downy mildew) Java downy mildew*Peronosclerospora maydis = Sclerospora maydis Philippine downyPeronosclerospora philippinensis = mildew* Sclerospora philippinensisSorghum downy mildew Peronosclerospora sorghi = Sclerospora sorghiSpontaneum downy Peronosclerospora spontanea = Sclerospora mildew*spontanea Sugarcane downy Peronosclerospora sacchari = Sclerosporamildew* sacchari Dry ear rot (cob, Nigrospora oryzae (teleomorph:Khuskia kernel and stalk rot) oryzae) Ear rots, minor Alternariaalternata = A. tenuis, Aspergillus glaucus, A. niger, Aspergillus spp.,Botrytis cinerea (teleomorph: Botryotinia fuckeliana), Cunninghamellasp., Curvularia pallescens, Doratomyces stemonitis = Cephalotrichumstemonitis, Fusarium culmorum, Gonatobotrys simplex, Pithomycesmaydicus, Rhizopus microsporus Tiegh., R. stolonifer = R. nigricans,Scopulariopsis brumptii. Ergot* (horse's tooth, Claviceps gigantea(anamorph: Sphacelia sp.) diente de caballo) Eyespot Aureobasidium zeae= Kabatiella zeae Fusarium ear and Fusarium subglutinans = F.moniliforme var. stalk rot subglutinans Fusarium kernel, root Fusariummoniliforme (teleomorph: Gibberella and stalk rot, seed rot fujikuroi)and seedling blight Fusarium stalk rot, Fusarium avenaceum (teleomorph:Gibberlla seedling root rot avenacea) Gibberella ear and Gibberella zeae(anamorph: Fusarium stalk rot graminearum) Gray ear rot Botryosphaeriazeae = Physalospora zeae (anamorph: Macrophoma zeae) Gray leaf spotCercospora sorghi = C. sorghi var. maydis, C. (Cercospora leaf spot)zeae-maydis Helminthosporium root Exserohilum pedicellatum =Helminthosporium rot pedicellatum (teleomorph: Setosphaeria pedicellata)Hormodendrum ear rot Cladosporium cladosporioides = (Cladosporium rot)Hormodendrum cladosporioides, C. herbarum (teleomorph: Mycosphaerellatassiana) Hyalothyridium leaf Hyalothyridium maydis spot* Late wilt*Cephalosporium maydis Leaf spots, minor Alternaria alternata, Ascochytamaydis, A. tritici, A. zeicola, Bipolaris victoriae = Helminthosporiumvictoriae (teleomorph: Cochliobolus victoriae), C. sativus (anamorph:Bipolaris sorokiniana = H. sorokinianum = H. sativum), Epicoccum nigrum,Exserohilum prolatum = Drechslera prolata (teleomorph: Setosphaeriaprolata) Graphium penicillioides, Leptosphaeria maydis, Leptothyriumzeae, Ophiosphaerella herpotricha, (anamorph: Scolecosporiella sp.),Paraphaeosphaeria michotii, Phoma sp., Septoria zeae, S. zeicola, S.zeina Northern corn leaf Setosphaeria turcica (anarnorph: Exserohilumblight (white blast, crown turcicum = Helminthosporium turcicum) stalkrot, stripe) Northern corn leaf spot Cochliobolus carbonum (anamorph:Bipolaris Helminthosporium ear zeicola = Helminthosporium carbonum) rot(race 1) Penicillium ear rot (blue Penicillium spp., P. chrysogenum, P.eye, blue mold) expansum, P. oxalicum Phaeocytostroma stalkPhaeocytostroma ambiguum, = rot and root rot Phaeocytosporella zeaePhaeosphaeria leaf spot* Phaeosphaeria maydis = Sphaerulina maydisPhysalospora ear rot Botryosphaeria festucae = Physalospora(Botryosphaeria ear rot) zeicola (anamorph: Diplodia frumenti) Purpleleaf sheath Hemiparasitic bacteria and fungi Pyrenochaeta stalk rotPhoma terrestris = Pyrenochaeta terrestris and root rot Pythium root rotPythium spp., P. arrhenomanes, P. graminicola Pythium stalk rot Pythiumaphanidermatum = P. butleri L. Red kernel disease (ear Epicoccum nigrummold, leaf and seed rot) Rhizoctonia ear rot Rhizoctonia zeae(teleomorph: Waitea (sclerotial rot) circinata) Rhizoctonia root rot andRhizoctonia solani, Rhizoctonia zeae stalk rot Root rots, minorAlternaria alternata, Cercospora sorghi, Dictochaeta fertilis, Fusariumacuminatum (teleomorph: Gibberella acuminata), F. equiseti (teleomorph:G. intricans), F. oxysporum, F. pallidoroseum, F. poae, F. roseum, G.cyanogena, (anamorph: F. sulphureum), Microdochium bolleyi, Mucor sp.,Periconia circinata, Phytophthora cactorum, P. drechsleri, P. nicotianaevar. parasitica, Rhizopus arrhizus Rostratum leaf spot Setosphaeriarostrata, (anamorph: (Helminthosporium leaf Exserohilum rostratum =He/minthosporium disease, ear and stalk rot) rostratum) Rust, commoncorn Puccinia sorghi Rust, southern corn Puccinia polysora Rust,tropical corn Physopella pallescens, P. zeae = Angiopsora zeaeSclerotium ear rot* Sclerotium rolfsii Sacc. (teleomorph: Athelia(southern blight) rolfsii) Seed rot-seedling blight Bipolarissorokiniana, B. zeicola = Helminthosporium carbonum, Diplodia maydis,Exserohilum pedicillatum, Exserohilum turcicum = Helminthosporiumturcicum, Fusarium avenaceum, F. culmorum, F. moniliforme, Gibberellazeae (anamorph: F. graminearum), Macrophomina phaseolina, Penicilliumspp., Phomopsis sp., Pythium spp., Rhizoctonia solani, R. zeae,Sclerotium rolfsii, Spicaria sp. Selenophoma leaf spot* Selenophoma sp.Sheath rot Gaeumannomyces graminis Shuck rot Myrothecium gramineumSilage mold Monascus purpureus, M ruber Smut, common Ustilago zeae = U.maydis) Smut, false Ustilaginoidea virens Smut, head Sphacelothecareiliana = Sporisorium holci- sorghi Southern corn leaf blightCochliobolus heterostrophus (anamorph: and stalk rot Bipolaris maydis =Helminthosporium maydis) Southern leaf spot Stenocarpella macrospora =Diplodia macrospora Stalk rots, minor Cercospora sorghi, Fusariumepisphaeria, F. merismoides, F. oxysporum Schlechtend, F. poae, F.roseum, F. solani (teleomorph: Nectria haematococca), F. tricinctum,Mariannaea elegans, Mucor sp., Rhopographus zeae, Spicaria sp. Storagerots Aspergillus spp., Penicillium spp. and other fungi Tar spot*Phyllachora maydis Trichoderma ear rot Trichoderma viride = T. lignorumteleomorph: and root rot Hypocrea sp. White ear rot, root Stenocarpellamaydis = Diplodia zeae and stalk rot Yellow leaf blight Ascochytaischaemi, Phyllosticta maydis (teleomorph: Mycosphaerella zeae-maydis)Zonate leaf spot Gloeocercospora sorghi *Not known to occur naturally oncorn in the United States.

Plant parasitic nematodes are a cause of disease in many plants,including maize. It is proposed that it would be possible to make plantsresistant to these organisms through the expression of novel geneproducts. It is anticipated that control of nematode infestations wouldbe accomplished by altering the ability of the nematode to recognize orattach to a host plant and/or enabling the plant to produce nematicidalcompounds, including but not limited to proteins. Examples ofnematode-associated plant diseases, for which one could introduceresistance to in a transgenic plant in accordance with the invention aregiven below, in Table 6.

TABLE 6 Parasitic Nematodes DISEASE PATHOGEN Awl Dolichodorus spp., D.heterocephalus Bulb and stem (Europe) Ditylenchus dipsaci BurrowingRadopholus similis Cyst Heterodera avenae, H. zeae, Punctoderachalcoensis Dagger Xiphinema spp., X. americanum, X. mediterraneum Falseroot-knot Nacobbus dorsalis Lance, Columbia Hoplolaimus columbus LanceHoplolaimus spp., H. galeatus Lesion Pratylenchus spp., P. brachyurus,P. crenatus, P. hexincisus, P. neglectus, P. penetrans, P. scribneri, P.thornei, P. zeae Needle Longidorus spp., L. breviannulatus RingCriconemella spp., C. ornata Root-knot Meloidogyne spp., M. chitwoodi,M. incognita, M. javanica Spiral Helicotylenchus spp. Sting Belonolaimusspp., B. longicaudatus Stubby-root Paratrichodorus spp., P. christiei,P. minor, Quinisulcius acutus, Trichodorus spp. Stunt Tylenchorhynchusdubius

(v) Mycotoxin Reduction/Elimination

Production of mycotoxins, including aflatoxin and fumonisin, by fungiassociated with monocotyledonous plants such as maize is a significantfactor in rendering the grain not useful. These fungal organisms do notcause disease symptoms and/or interfere with the growth of the plant,but they produce chemicals (mycotoxins) that are toxic to animals. It iscontemplated that inhibition of the growth of these fungi would reducethe synthesis of these toxic substances and therefore reduce grainlosses due to mycotoxin contamination. It also is proposed that it maybe possible to introduce novel genes into monocotyledonous plants suchas maize that would inhibit synthesis of the mycotoxin. Further, it iscontemplated that expression of a novel gene which encodes an enzymecapable of rendering the mycotoxin nontoxic would be useful in order toachieve reduced mycotoxin contamination of grain. The result of any ofthe above mechanisms would be a reduced presence of mycotoxins on grain.

(vi) Grain Composition or Quality

Genes may be introduced into monocotyledonous plants, particularlycommercially important cereals such as maize, to improve the grain forwhich the cereal is primarily grown. A wide range of novel transgenicplants produced in this manner may be envisioned depending on theparticular end use of the grain.

The largest use of maize grain is for feed or food. Introduction ofgenes that alter the composition of the grain may greatly enhance thefeed or food value. The primary components of maize grain are starch,protein, and oil. Each of these primary components of maize grain may beimproved by altering its level or composition. Several examples may bementioned for illustrative purposes, but in no way provide an exhaustivelist of possibilities.

The protein of cereal grains including maize is suboptimal for feed andfood purposes especially when fed to pigs, poultry, and humans. Theprotein is deficient in several amino acids that are essential in thediet of these species, requiring the addition of supplements to thegrain. Limiting essential amino acids may include lysine, methionine,tryptophan, threonine, valine, arginine, and histidine. Some amino acidsbecome limiting only after corn is supplemented with other inputs forfeed formulations. For example, when corn is supplemented with soybeanmeal to meet lysine requirements methionine becomes limiting. The levelsof these essential amino acids in seeds and grain may be elevated bymechanisms which include, but are not limited to, the introduction ofgenes to increase the biosynthesis of the amino acids, decrease thedegradation of the amino acids, increase the storage of the amino acidsin proteins, or increase transport of the amino acids to the seeds orgrain.

One mechanism for increasing the biosynthesis of the amino acids is tointroduce genes that deregulate the amino acid biosynthetic pathwayssuch that the plant can no longer adequately control the levels that areproduced. This may be done by deregulating or bypassing steps in theamino acid biosynthetic pathway which are normally regulated by levelsof the amino acid end product of the pathway. Examples include theintroduction of genes that encode deregulated versions of the enzymesaspartokinase or dihydrodipicolinic acid (DHDP)-synthase for increasinglysine and threonine production, and anthranilate synthase forincreasing tryptophan production. Reduction of the catabolism of theamino acids may be accomplished by introduction of DNA sequences thatreduce or eliminate the expression of genes encoding enzymes thatcatalyze steps in the catabolic pathways such as the enzymelysine-ketoglutarate reductase. It is anticipated that it may bedesirable to target expression of genes relating to amino acidbiosynthesis to the endosperm or embryo of the seed. More preferably,the gene will be targeted to the embryo. It will also be preferable forgenes encoding proteins involved in amino acid biosynthesis to targetthe protein to a plastid using a plastid transit peptide sequence.

The protein composition of the grain may be altered to improve thebalance of amino acids in a variety of ways including elevatingexpression of native proteins, decreasing expression of those with poorcomposition, changing the composition of native proteins, or introducinggenes encoding entirely new proteins possessing superior composition.Examples may include the introduction of DNA that decreases theexpression of members of the zein family of storage proteins. This DNAmay encode ribozymes or antisense sequences directed to impairingexpression of zein proteins or expression of regulators of zeinexpression such as the opaque-2 gene product. It also is proposed thatthe protein composition of the grain may be modified through thephenomenon of co-suppression, i.e., inhibition of expression of anendogenous gene through the expression of an identical structural geneor gene fragment introduced through transformation (Goring et al.,1991). Additionally, the introduced DNA may encode enzymes which degradezeins. The decreases in zein expression that are achieved may beaccompanied by increases in proteins with more desirable amino acidcomposition or increases in other major seed constituents such asstarch. Alternatively, a chimeric gene may be introduced that comprisesa coding sequence for a native protein of adequate amino acidcomposition such as for one of the globulin proteins or 10 kD delta zeinor 20 kD delta zein or 27 kD gamma zein of maize and a promoter or otherregulatory sequence designed to elevate expression of said protein. Thecoding sequence of the gene may include additional or replacement codonsfor essential amino acids. Further, a coding sequence obtained fromanother species, or, a partially or completely synthetic sequenceencoding a completely unique peptide sequence designed to enhance theamino acid composition of the seed may be employed. It is anticipatedthat it may be preferable to target expression of these transgenesencoding proteins with superior composition to the endosperm of theseed.

The introduction of genes that alter the oil content of the grain may beof value. Increases in oil content may result in increases inmetabolizable-energy-content and density of the seeds for use in feedand food. The introduced genes may encode enzymes that remove or reducerate-limitations or regulated steps in fatty acid or lipid biosynthesis.Such genes may include, but are not limited to, those that encodeacetyl-CoA carboxylase, ACP-acyltransferase, β-ketoacyl-ACP synthase,plus other well known fatty acid biosynthetic activities. Otherpossibilities are genes that encode proteins that do not possessenzymatic activity such as acyl carrier protein. Genes may be introducedthat alter the balance of fatty acids present in the oil providing amore healthful or nutritive feedstuff. The introduced DNA also mayencode sequences that block expression of enzymes involved in fatty acidbiosynthesis, altering the proportions of fatty acids present in thegrain such as described below. Some other examples of genes specificallycontemplated by the inventors for use in creating transgenic plants withaltered oil composition traits include 2-acetyltransferase, oleosin,pyruvate dehydrogenase complex, acetyl CoA synthetase, ATP citratelyase, ADP-glucose pyrophosphorylase and genes of thecarnitine-CoA-acetyl-CoA shuttles. It is anticipated that expression ofgenes related to oil biosynthesis will be targeted to the plastid, usinga plastid transit peptide sequence and preferably expressed in the seedembryo.

Genes may be introduced that enhance the nutritive value of the starchcomponent of the grain, for example by increasing the degree ofbranching, resulting in improved utilization of the starch in cows bydelaying its metabolism. It is anticipated that expression of genesrelated to starch biosynthesis will preferably be targeted to theendosperm of the seed.

Besides affecting the major constituents of the grain, genes may beintroduced that affect a variety of other nutritive, processing, orother quality aspects of the grain as used for feed or food. Forexample, pigmentation of the grain may be increased or decreased.Enhancement and stability of yellow pigmentation is desirable in someanimal feeds and may be achieved by introduction of genes that result inenhanced production of xanthophylls and carotenes by eliminatingrate-limiting steps in their production. Such genes may encode alteredforms of the enzymes phytoene synthase, phytoene desaturase, or lycopenesynthase. Alternatively, unpigmented white corn is desirable forproduction of many food products and may be produced by the introductionof DNA which blocks or eliminates steps in pigment production pathways.

Most of the phosphorous content of the grain is in the form of phytate,a form of phosphate storage that is not metabolized by monogastricanimals. Therefore, in order to increase the availability of seedphosphate, it is anticipated that one will desire to decrease the amountof phytate in seed and increase the amount of free phosphorous. It isanticipated that one can decrease the expression or activity of one ofthe enzymes involved in the synthesis of phytate. For example,suppression of expression of the gene encoding inositol phosphatesynthetase (INOPS) may lead to an overall reduction in phytateaccumulation. It is anticipated that antisense or sense suppression ofgene expression may be used. Alternatively, one may express a gene incorn seed which will be activated, e.g., by pH, in the gastric system ofa monogastric animal and will release phosphate from phytate. e.g.,phytase.

Feed or food comprising primarily maize or other cereal grains possessesinsufficient quantities of vitamins and must be supplemented to provideadequate nutritive value. Introduction of genes that enhance vitaminbiosynthesis in seeds may be envisioned including, for example, vitaminsA, E, B₁₂, choline, and the like. Maize grain also does not possesssufficient mineral content for optimal nutritive value. Genes thataffect the accumulation or availability of compounds containingphosphorus, sulfur, calcium, manganese, zinc, and iron among otherswould be valuable. An example may be the introduction of a gene thatreduced phytic acid production or encoded the enzyme phytase whichenhances phytic acid breakdown. These genes would increase levels ofavailable phosphate in the diet, reducing the need for supplementationwith mineral phosphate.

Numerous other examples of improvement of maize or other cereals forfeed and food purposes might be described. The improvements may not evennecessarily involve the grain, but may, for example, improve the valueof the corn for silage. Introduction of DNA to accomplish this mightinclude sequences that alter lignin production such as those that resultin the “brown midrib” phenotype associated with superior feed value forcattle.

In addition to direct improvements in feed or food value, genes also maybe introduced which improve the processing of corn and improve the valueof the products resulting from the processing. The primary method ofprocessing corn is via wetmilling. Maize may be improved though theexpression of novel genes that increase the efficiency and reduce thecost of processing such as by decreasing steeping time.

Improving the value of wetmilling products may include altering thequantity or quality of starch, oil, corn gluten meal, or the componentsof corn gluten feed. Elevation of starch may be achieved through theidentification and elimination of rate limiting steps in starchbiosynthesis or by decreasing levels of the other components of thegrain resulting in proportional increases in starch. An example of theformer may be the introduction of genes encoding ADP-glucosepyrophosphorylase enzymes with altered regulatory activity or which areexpressed at higher level. Examples of the latter may include selectiveinhibitors of, for example, protein or oil biosynthesis expressed duringlater stages of kernel development.

The properties of starch may be beneficially altered by changing theratio of amylose to amylopectin, the size of the starch molecules, ortheir branching pattern. Through these changes a broad range ofproperties may be modified which include, but are not limited to,changes in gelatinization temperature, heat of gelatinization, clarityof films and pastes, rheological properties, and the like. To accomplishthese changes in properties, genes that encode granule-bound or solublestarch synthase activity or branching enzyme activity may be introducedalone or combination. DNA such as antisense constructs also may be usedto decrease levels of endogenous activity of these enzymes. Theintroduced genes or constructs may possess regulatory sequences thattime their expression to specific intervals in starch biosynthesis andstarch granule development. Furthermore, it may be worthwhile tointroduce and express genes that result in the in vivo derivatization,or other modification, of the glucose moieties of the starch molecule.The covalent attachment of any molecule may be envisioned, limited onlyby the existence of enzymes that catalyze the derivatizations and theaccessibility of appropriate substrates in the starch granule. Examplesof important derivations may include the addition of functional groupssuch as amines, carboxyls, or phosphate groups which provide sites forsubsequent in vitro derivatizations or affect starch properties throughthe introduction of ionic charges. Examples of other modifications mayinclude direct changes of the glucose units such as loss of hydroxylgroups or their oxidation to aldehyde or carboxyl groups.

Oil is another product of wetmilling of corn, the value of which may beimproved by introduction and expression of genes. The quantity of oilthat can be extracted by wetmilling may be elevated by approaches asdescribed for feed and food above. Oil properties also may be altered toimprove its performance in the production and use of cooking oil,shortenings, lubricants or other oil-derived products or improvement ofits health attributes when used in the food-related applications. Novelfatty acids also may be synthesized which upon extraction can serve asstarting materials for chemical syntheses. The changes in oil propertiesmay be achieved by altering the type, level, or lipid arrangement of thefatty acids present in the oil. This in turn may be accomplished by theaddition of genes that encode enzymes that catalyze the synthesis ofnovel fatty acids and the lipids possessing them or by increasing levelsof native fatty acids while possibly reducing levels of precursors.Alternatively, DNA sequences may be introduced which slow or block stepsin fatty acid biosynthesis resulting in the increase in precursor fattyacid intermediates. Genes that might be added include desaturases,epoxidases, hydratases, dehydratases, and other enzymes that catalyzereactions involving fatty acid intermediates. Representative examples ofcatalytic steps that might be blocked include the desaturations fromstearic to oleic acid and oleic to linolenic acid resulting in therespective accumulations of stearic and oleic acids. Another example isthe blockage of elongation steps resulting in the accumulation of C₈ toC₁₂ saturated fatty acids.

Improvements in the other major corn wetmilling products, corn glutenmeal and corn gluten feed, also may be achieved by the introduction ofgenes to obtain novel corn plants. Representative possibilities includebut are not limited to those described above for improvement of food andfeed value.

In addition, it may further be considered that the corn plant be usedfor the production or manufacturing of useful biological compounds thatwere either not produced at all, or not produced at the same level, inthe corn plant previously. The novel corn plants producing thesecompounds are made possible by the introduction and expression of genesby corn transformation methods. The vast array of possibilities includebut are not limited to any biological compound which is presentlyproduced by any organism such as proteins, nucleic acids, primary andintermediary metabolites, carbohydrate polymers, etc. The compounds maybe produced by the plant, extracted upon harvest and/or processing, andused for any presently recognized useful purpose such aspharmaceuticals, fragrances, and industrial enzymes to name a few.

Further possibilities to exemplify the range of grain traits orproperties potentially encoded by introduced genes in transgenic plantsinclude grain with less breakage susceptibility for export purposes orlarger grit size when processed by dry milling through introduction ofgenes that enhance γ-zein synthesis, popcorn with improved poppingquality and expansion volume through genes that increase pericarpthickness, corn with whiter grain for food uses though introduction ofgenes that effectively block expression of enzymes involved in pigmentproduction pathways, and improved quality of alcoholic beverages orsweet corn through introduction of genes which affect flavor such as theshrunken 1 gene (encoding sucrose synthase) or shrunken 2 gene (encodingADPG pyrophosphorylase) for sweet corn.

(vii) Plant Agronomic Characteristics

Two of the factors determining where crop plants can be grown are theaverage daily temperature during the growing season and the length oftime between frosts. Within the areas where it is possible to grow aparticular crop, there are varying limitations on the maximal time it isallowed to grow to maturity and be harvested. For example, maize to begrown in a particular area is selected for its ability to mature and drydown to harvestable moisture content within the required period of timewith maximum possible yield. Therefore, corn of varying maturities isdeveloped for different growing locations. Apart from the need to drydown sufficiently to permit harvest, it is desirable to have maximaldrying take place in the field to minimize the amount of energy requiredfor additional drying post-harvest. Also, the more readily the grain candry down, the more time there is available for growth and kernel fill.It is considered that genes that influence maturity and/or dry down canbe identified and introduced into corn or other plants usingtransformation techniques to create new varieties adapted to differentgrowing locations or the same growing location, but having improvedyield to moisture ratio at harvest. Expression of genes that areinvolved in regulation of plant development may be especially useful,e.g., the liguleless and rough sheath genes that have been identified incorn.

It is contemplated that genes may be introduced into plants that wouldimprove standability and other plant growth characteristics. Expressionof novel genes in maize which confer stronger stalks, improved rootsystems, or prevent or reduce ear droppage would be of great value tothe farmer. It is proposed that introduction and expression of genesthat increase the total amount of photoassimilate available by, forexample, increasing light distribution and/or interception would beadvantageous. In addition, the expression of genes that increase theefficiency of photosynthesis and/or the leaf canopy would furtherincrease gains in productivity. It is contemplated that expression of aphytochrome gene in corn may be advantageous. Expression of such a genemay reduce apical dominance, confer semidwarfism on a plant, andincrease shade tolerance (U.S. Pat. No. 5,268,526). Such approacheswould allow for increased plant populations in the field.

Delay of late season vegetative senescence would increase the flow ofassimilate into the grain and thus increase yield. It is proposed thatoverexpression of genes within corn that are associated with “staygreen” or the expression of any gene that delays senescence would beadvantageous. For example, a nonyellowing mutant has been identified inFestuca pratensis (Davies et al., 1990). Expression of this gene as wellas others may prevent premature breakdown of chlorophyll and thusmaintain canopy function.

(viii) Nutrient Utilization

The ability to utilize available nutrients may be a limiting factor ingrowth of monocotyledonous plants such as maize. It is proposed that itwould be possible to alter nutrient uptake, tolerate pH extremes,mobilization through the plant, storage pools, and availability formetabolic activities by the introduction of novel genes. Thesemodifications would allow a plant such as maize to more efficientlyutilize available nutrients. It is contemplated that an increase in theactivity of, for example, an enzyme that is normally present in theplant and involved in nutrient utilization would increase theavailability of a nutrient. An example of such an enzyme would bephytase. It further is contemplated that enhanced nitrogen utilizationby a plant is desirable. Expression of a glutamate dehydrogenase gene incorn, e.g., E. coli gdhA genes, may lead to increased fixation ofnitrogen in organic compounds. Furthermore, expression of gdhA in cornmay lead to enhanced resistance to the herbicide glufosinate byincorporation of excess ammonia into glutamate, thereby detoxifying theammonia. It also is contemplated that expression of a novel gene maymake a nutrient source available that was previously not accessible,e.g., an enzyme that releases a component of nutrient value from a morecomplex molecule, perhaps a macromolecule.

(ix) Male Sterility

Male sterility is useful in the production of hybrid seed. It isproposed that male sterility may be produced through expression of novelgenes. For example, it has been shown that expression of genes thatencode proteins that interfere with development of the maleinflorescence and/or gametophyte result in male sterility. Chimericribonuclease genes that express in the anthers of transgenic tobacco andoilseed rape have been demonstrated to lead to male sterility (Marianiet al., 1990).

A number of mutations were discovered in maize that confer cytoplasmicmale sterility. One mutation in particular, referred to as T cytoplasm,also correlates with sensitivity to Southern corn leaf blight. A DNAsequence, designated TURF-13 (Levings, 1990), was identified thatcorrelates with T cytoplasm. It is proposed that it would be possiblethrough the introduction of TURF-13 via transformation, to separate malesterility from disease sensitivity. As it is necessary to be able torestore male fertility for breeding purposes and for grain production,it is proposed that genes encoding restoration of male fertility alsomay be introduced.

(x) Negative Selectable Markers

Introduction of genes encoding traits that can be selected against maybe useful for eliminating undesirable linked genes. It is contemplatedthat when two or more genes are introduced together by cotransformationthat the genes will be linked together on the host chromosome. Forexample, a gene encoding Bt that confers insect resistance on the plantmay be introduced into a plant together with a bar gene that is usefulas a selectable marker and confers resistance to the herbicide Liberty®on the plant. However, it may not be desirable to have an insectresistant plant that also is resistant to the herbicide Liberty®. It isproposed that one also could introduce an antisense bar coding regionthat is expressed in those tissues where one does not want expression ofthe bar gene product, e.g., in whole plant parts. Hence, although thebar gene is expressed and is useful as a selectable marker, it is notuseful to confer herbicide resistance on the whole plant. The barantisense gene is a negative selectable marker.

It also is contemplated that negative selection is necessary in order toscreen a population of transformants for rare homologous recombinantsgenerated through gene targeting. For example, a homologous recombinantmay be identified through the inactivation of a gene that was previouslyexpressed in that cell. The antisense construct for neomycinphosphotransferase II (NPT II) has been investigated as a negativeselectable marker in tobacco (Nicotiana tabacum) and Arabidopsisthaliana (Xiang. and Guerra, 1993). In this example, both sense andantisense NPT II genes are introduced into a plant throughtransformation and the resultant plants are sensitive to the antibiotickanamycin. An introduced gene that integrates into the host cellchromosome at the site of the antisense NPT II gene, and inactivates theantisense gene, will make the plant resistant to kanamycin and otheraminoglycoside antibiotics. Therefore, rare, site-specific recombinantsmay be identified by screening for antibiotic resistance. Similarly, anygene, native to the plant or introduced through transformation, thatwhen inactivated confers resistance to a compound, may be useful as anegative selectable marker.

It is contemplated that negative selectable markers also may be usefulin other ways. One application is to construct transgenic lines in whichone could select for transposition to unlinked sites. In the process oftagging it is most common for the transposable element to move to agenetically linked site on the same chromosome. A selectable marker forrecovery of rare plants in which transposition has occurred to anunlinked locus would be useful. For example, the enzyme cytosinedeaminase may be useful for this purpose (Stouggard, 1993). In thepresence of this enzyme the compound 5-fluorocytosine is converted to5-fluorouracil which is toxic to plant and animal cells. If atransposable element is linked to the gene for the enzyme cytosinedeaminase, one may select for transposition to unlinked sites byselecting for transposition events in which the resultant plant is nowresistant to 5-fluorocytosine. The parental plants and plants containingtranspositions to linked sites will remain sensitive to5-fluorocytosine. Resistance to 5-fluorocytosine is due to loss of thecytosine deaminase gene through genetic segregation of the transposableelement and the cytosine deaminase gene. Other genes that encodeproteins that render the plant sensitive to a certain compound will alsobe useful in this context. For example, T-DNA gene 2 from Agrobacteriumtumefaciens encodes a protein that catalyzes the conversion ofα-naphthalene acetamide (NAM) to α-naphthalene acetic acid (NAA) rendersplant cells sensitive to high concentrations of NAM (Depicker et al.,1988).

It also is contemplated that negative selectable markers may be usefulin the construction of transposon tagging lines. For example, by markingan autonomous transposable element such as Ac, Master Mu, or En/Spn witha negative selectable marker, one could select for transformants inwhich the autonomous element is not stably integrated into the genome.It is proposed that this would be desirable, for example, when transientexpression of the autonomous element is desired to activate in trans thetransposition of a defective transposable element, such as Ds, butstable integration of the autonomous element is not desired. Thepresence of the autonomous element may not be desired in order tostabilize the defective element, i.e., prevent it from furthertransposing. However, it is proposed that if stable integration of anautonomous transposable element is desired in a plant the presence of anegative selectable marker may make it possible to eliminate theautonomous element during the breeding process.

(xi) Non-Protein-Expressing Sequences

DNA may be introduced into plants for the purpose of expressing RNAtranscripts that function to affect plant phenotype yet are nottranslated into protein. Two examples are antisense RNA and RNA withribozyme activity. Both may serve possible functions in reducing oreliminating expression of native or introduced plant genes. However, asdetailed below, DNA need not be expressed to effect the phenotype of aplant.

1. Antisense RNA

Genes may be constructed or isolated, which when transcribed, produceantisense RNA that is complementary to all or part(s) of a targetedmessenger RNA(s). The antisense RNA reduces production of thepolypeptide product of the messenger RNA. The polypeptide product may beany protein encoded by the plant genome. The aforementioned genes willbe referred to as antisense genes. An antisense gene may thus beintroduced into a plant by transformation methods to produce a noveltransgenic plant with reduced expression of a selected protein ofinterest. For example, the protein may be an enzyme that catalyzes areaction in the plant. Reduction of the enzyme activity may reduce oreliminate products of the reaction which include any enzymaticallysynthesized compound in the plant such as fatty acids, amino acids,carbohydrates, nucleic acids and the like. Alternatively, the proteinmay be a storage protein, such as a zein, or a structural protein, thedecreased expression of which may lead to changes in seed amino acidcomposition or plant morphological changes respectively. Thepossibilities cited above are provided only by way of example and do notrepresent the full range of applications.

2. Ribozymes

Genes also may be constructed or isolated, which when transcribed,produce RNA enzymes (ribozymes) which can act as endoribonucleases andcatalyze the cleavage of RNA molecules with selected sequences. Thecleavage of selected messenger RNAs can result in the reduced productionof their encoded polypeptide products. These genes may be used toprepare novel transgenic plants which possess them. The transgenicplants may possess reduced levels of polypeptides including, but notlimited to, the polypeptides cited above.

Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987;Forster and Symons, 1987). For example, a large number of ribozymesaccelerate phosphoester transfer reactions with a high degree ofspecificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes.

Several different ribozyme motifs have been described with RNA cleavageactivity (Symons, 1992). Examples include sequences from the Group Iself splicing introns including Tobacco Ringspot Virus (Prody et al.,1986), Avocado Sunblotch Viroid (Palukaitis et al., 1979; Symons, 1981),and Lucerne Transient Streak Virus (Forster and Symons, 1987). Sequencesfrom these and related viruses are referred to as hammerhead ribozymebased on a predicted folded secondary structure.

Other suitable ribozymes include sequences from RNase P with RNAcleavage activity (Yuan et al., 1992, Yuan and Altman, 1994, U.S. Pat.Nos. 5,168,053 and 5,624,824), hairpin ribozyme structures(Berzal-Herranz et al., 1992; Chowrira et al., 1993) and Hepatitis Deltavirus based ribozymes (U.S. Pat. No. 5,625,047). The general design andoptimization of ribozyme directed RNA cleavage activity has beendiscussed in detail (Haseloff and Gerlach, 1988, Symons, 1992, Chowriraet al., 1994; Thompson et al., 1995).

The other variable on ribozyme design is the selection of a cleavagesite on a given target RNA. Ribozymes are targeted to a given sequenceby virtue of annealing to a site by complimentary base pairinteractions. Two stretches of homology are required for this targeting.These stretches of homologous sequences flank the catalytic ribozymestructure defined above. Each stretch of homologous sequence can vary inlength from 7 to 15 nucleotides. The only requirement for defining thehomologous sequences is that, on the target RNA, they are separated by aspecific sequence which is the cleavage site. For hammerhead ribozyme,the cleavage site is a dinucleotide sequence on the target RNA is auracil (U) followed by either an adenine, cytosine or uracil (A,C or U)(Perriman et al., 1992; Thompson et al., 1995). The frequency of thisdinucleotide occurring in any given RNA is statistically 3 out of 16.Therefore, for a given target messenger RNA of 1000 bases, 187dinucleotide cleavage sites are statistically possible.

Designing and testing ribozymes for efficient cleavage of a target RNAis a process well known to those skilled in the art. Examples ofscientific methods for designing and testing ribozymes are described byChowrira et al., (1994) and Lieber and Strauss (1995), each incorporatedby reference. The identification of operative and preferred sequencesfor use in down regulating a given gene is simply a matter of preparingand testing a given sequence, and is a routinely practiced “screening”method known to those of skill in the art.

3. Induction of Gene Silencing

It also is possible that genes may be introduced to produce noveltransgenic plants which have reduced expression of a native gene productby the mechanism of co-suppression. It has been demonstrated in tobacco,tomato, and petunia (Goring et al., 1991; Smith et al., 1990; Napoli etal., 1990; van der Krol et al., 1990) that expression of the sensetranscript of a native gene will reduce or eliminate expression of thenative gene in a manner similar to that observed for antisense genes.The introduced gene may encode all or part of the targeted nativeprotein but its translation may not be required for reduction of levelsof that native protein.

4. Non-RNA-Expressing Sequences

DNA elements including those of transposable elements such as Ds, Ac, orMu, may be inserted into a gene to cause mutations. These DNA elementsmay be inserted in order to inactivate (or activate) a gene and thereby“tag” a particular trait. In this instance the transposable element doesnot cause instability of the tagged mutation, because the utility of theelement does not depend on its ability to move in the genome. Once adesired trait is tagged, the introduced DNA sequence may be used toclone the corresponding gene, e.g., using the introduced DNA sequence asa PCR primer together with PCR gene cloning techniques (Shapiro, 1983;Dellaporta et al., 1988). Once identified, the entire gene(s) for theparticular trait, including control or regulatory regions where desired,may be isolated, cloned and manipulated as desired. The utility of DNAelements introduced into an organism for purposes of gene tagging isindependent of the DNA sequence and does not depend on any biologicalactivity of the DNA sequence, i.e., transcription into RNA ortranslation into protein. The sole function of the DNA element is todisrupt the DNA sequence of a gene.

It is contemplated that unexpressed DNA sequences, including novelsynthetic sequences, could be introduced into cells as proprietary“labels” of those cells and plants and seeds thereof. It would not benecessary for a label DNA element to disrupt the function of a geneendogenous to the host organism, as the sole function of this DNA wouldbe to identify the origin of the organism. For example, one couldintroduce a unique DNA sequence into a plant and this DNA element wouldidentify all cells, plants, and progeny of these cells as having arisenfrom that labeled source. It is proposed that inclusion of label DNAswould enable one to distinguish proprietary germplasm or germplasmderived from such, from unlabelled germplasm.

Another possible element which may be introduced is a matrix attachmentregion element (MAR), such as the chicken lysozyme A element (Stief,1989), which can be positioned around an expressible gene of interest toeffect an increase in overall expression of the gene and diminishposition dependent effects upon incorporation into the plant genome(Stief et al., 1989; Phi-Van et al., 1990).

IV. Assays of Transgene Expression

Assays may be employed with the instant invention for determination ofthe relative efficiency of transgene expression. For example, assays maybe used to determine the efficacy of deletion mutants of the RS81promoter in directing expression of exogenous proteins. Similarly, onecould produce random or site-specific mutants of the RS81 promoter ofthe invention and assay the efficacy of the mutants in the expression ofa given transgene. Alternatively, assays could be used to determine theefficacy of the RS81 promoter in directing protein expression when usedin conjunction with various different enhancers, terminators or othertypes of elements potentially used in the preparation of transformationconstructs.

For plants, expression assays may comprise a system utilizingembryogenic or non-embryogenic cells, or alternatively, whole plants. Anadvantage of using cellular assays is that regeneration of large numbersof plants is not required. However, the systems are limited in thatpromoter activity in the non-regenerated cells may not directlycorrelate with expression in a plant. Additionally, assays of tissue ordevelopmental specific promoters are generally not feasible.

The biological sample to be assayed may comprise nucleic acids isolatedfrom the cells of any plant material according to standard methodologies(Sambrook et al., 1989). The nucleic acid may be genomic DNA orfractionated or whole cell RNA. Where RNA is used, it may be desired toconvert the RNA to a complementary DNA. In one embodiment of theinvention, the RNA is whole cell RNA; in another, it is poly-A RNA.Normally, the nucleic acid is amplified.

Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g., ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology; Bellus, 1994).

Following detection, one may compare the results seen in a given plantwith a statistically significant reference group of non-transformedcontrol plants. Typically, the non-transformed control plants will be ofa genetic background similar to the transformed plants. In this way, itis possible to detect differences in the amount or kind of proteindetected in various transformed plants. Alternatively, clonal culturesof cells, for example, callus or an immature embryo, may be compared toother cells samples.

As indicated, a variety of different assays are contemplated in thescreening of cells or plants of the current invention and associatedpromoters. These techniques may in cases be used to detect for both thepresence and expression of the particular genes as well asrearrangements that may have occurred in the gene construct. Thetechniques include but are not limited to, fluorescent in situhybridization (FISH), direct DNA sequencing, pulsed field gelelectrophoresis (PFGE) analysis, Southern or Northern blotting,single-stranded conformation analysis (SSCA), RNAse protection assay,allele-specific oligonucleotide (ASO), dot blot analysis, denaturinggradient gel electrophoresis, RFLP and PCR™-SSCP.

(i) Quantitation of Gene Expression with Relative Quantitative RT-PCR™

Reverse transcription (RT) of RNA to cDNA followed by relativequantitative PCR™ (RT-PCR™) can be used to determine the relativeconcentrations of specific mRNA species isolated from plants. Bydetermining that the concentration of a specific mRNA species varies, itis shown that the gene encoding the specific mRNA species isdifferentially expressed. In this way, a promoters expression profilecan be rapidly identified, as can the efficacy with which the promoterdirects transgene expression.

In PCR™, the number of molecules of the amplified target DNA increase bya factor approaching two with every cycle of the reaction until somereagent becomes limiting. Thereafter, the rate of amplification becomesincreasingly diminished until there is no increase in the amplifiedtarget between cycles. If a graph is plotted in which the cycle numberis on the X axis and the log of the concentration of the amplifiedtarget DNA is on the Y axis, a curved line of characteristic shape isformed by connecting the plotted points. Beginning with the first cycle,the slope of the line is positive and constant. This is said to be thelinear portion of the curve. After a reagent becomes limiting, the slopeof the line begins to decrease and eventually becomes zero. At thispoint the concentration of the amplified target DNA becomes asymptoticto some fixed value. This is said to be the plateau portion of thecurve.

The concentration of the target DNA in the linear portion of the PCR™amplification is directly proportional to the starting concentration ofthe target before the reaction began. By determining the concentrationof the amplified products of the target DNA in PCR™ reactions that havecompleted the same number of cycles and are in their linear ranges, itis possible to determine the relative concentrations of the specifictarget sequence in the original DNA mixture. If the DNA mixtures arecDNAs synthesized from RNAs isolated from different tissues or cells,the relative abundances of the specific mRNA from which the targetsequence was derived can be determined for the respective tissues orcells. This direct proportionality between the concentration of the PCR™products and the relative mRNA abundances is only true in the linearrange of the PCR™ reaction.

The final concentration of the target DNA in the plateau portion of thecurve is determined by the availability of reagents in the reaction mixand is independent of the original concentration of target DNA.Therefore, the first condition that must be met before the relativeabundances of a mRNA species can be determined by RT-PCR™ for acollection of RNA populations is that the concentrations of theamplified PCR™ products must be sampled when the PCR™ reactions are inthe linear portion of their curves.

The second condition that must be met for an RT-PCR™ study tosuccessfully determine the relative abundances of a particular mRNAspecies is that relative concentrations of the amplifiable cDNAs must benormalized to some independent standard. The goal of an RT-PCR™ study isto determine the abundance of a particular mRNA species relative to theaverage abundance of all mRNA species in the sample.

Most protocols for competitive PCR™ utilize internal PCR™ standards thatare approximately as abundant as the target. These strategies areeffective if the products of the PCR™ amplifications are sampled duringtheir linear phases. If the products are sampled when the reactions areapproaching the plateau phase, then the less abundant product becomesrelatively over represented. Comparisons of relative abundances made formany different RNA samples, such as is the case when examining RNAsamples for differential expression, become distorted in such a way asto make differences in relative abundances of RNAs appear less than theyactually are. This is not a significant problem if the internal standardis much more abundant than the target. If the internal standard is moreabundant than the target, then direct linear comparisons can be madebetween RNA samples.

The above discussion describes theoretical considerations for an RT-PCR™assay for plant tissue. The problems inherent in plant tissue samplesare that they are of variable quantity (making normalizationproblematic), and that they are of variable quality (necessitating theco-amplification of a reliable internal control, preferably of largersize than the target). Both of these problems are overcome if theRT-PCR™ is performed as a relative quantitative RT-PCR™ with an internalstandard in which the internal standard is an amplifiable cDNA fragmentthat is larger than the target cDNA fragment and in which the abundanceof the mRNA encoding the internal standard is roughly 5-100 fold higherthan the mRNA encoding the target. This assay measures relativeabundance, not absolute abundance of the respective mRNA species.

Other studies may be performed using a more conventional relativequantitative RT-PCR™ assay with an external standard protocol. Theseassays sample the PCR™ products in the linear portion of theiramplification curves. The number of PCR™ cycles that are optimal forsampling must be empirically determined for each target cDNA fragment.In addition, the reverse transcriptase products of each RNA populationisolated from the various tissue samples must be carefully normalizedfor equal concentrations of amplifiable cDNAs. This consideration isvery important since the assay measures absolute mRNA abundance.Absolute mRNA abundance can be used as a measure of differential geneexpression only in normalized samples. While empirical determination ofthe linear range of the amplification curve and normalization of cDNApreparations are tedious and time consuming processes, the resultingRT-PCR™ assays can be superior to those derived from the relativequantitative RT-PCR™ assay with an internal standard.

One reason for this advantage is that without the internalstandard/competitor, all of the reagents can be converted into a singlePCR™ product in the linear range of the amplification curve, thusincreasing the sensitivity of the assay. Another reason is that withonly one PCR™ product, display of the product on an electrophoretic gelor another display method becomes less complex, has less background andis easier to interpret.

(ii) Marker Gene Expression

Marker genes represent an efficient means for assaying the expression oftransgenes. Using, for example, a selectable marker gene, one couldquantitatively determine the resistance conferred upon a plant or plantcell by a construct comprising the selectable marker coding regionoperably linked to the promoter to be assayed, e.g., an RS81 promoter.Alternatively, various plant parts could be exposed to a selective agentand the relative resistance provided in these parts quantified, therebyproviding an estimate of the tissue specific expression of the promoter.

Screenable markers constitute another efficient means for quantifyingthe expression of a given transgene. Potentially any screenable markercould be expressed and the marker gene product quantified, therebyproviding an estimate of the efficiency with which the promoter directsexpression of the transgene. Quantification can readily be carried outusing either visual means, or, for example, a photon counting device.

A preferred screenable marker gene assay for use with the currentinvention constitutes the use of the screenable marker geneβ-glucuronidase (GUS). Detection of GUS activity can be performedhistochemically using 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc) asthe substrate for the GUS enzyme, yielding a blue precipitate inside ofcells containing GUS activity. This assay has been described in detail(Jefferson, 1987). The blue coloration can then be visually scored, andestimates of expression efficiency thereby provided. GUS activity alsocan be determined by immunoblot analysis or a fluorometric GUS specificactivity assay (Jefferson, 1987).

(iii) Purification and Assays of Proteins

One means for determining the efficiency with which a particulartransgene is expressed is to purify and quantify a polypeptide expressedby the transgene. Protein purification techniques are well known tothose of skill in the art. These techniques involve, at one level, thecrude fractionation of the cellular milieu to polypeptide andnon-polypeptide fractions. Having separated the polypeptide from otherproteins, the polypeptide of interest may be further purified usingchromatographic and electrophoretic techniques to achieve partial orcomplete purification (or purification to homogeneity). Analyticalmethods particularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography; polyacrylamidegel electrophoresis; and isoelectric focusing. A particularly efficientmethod of purifying peptides is fast protein liquid chromatography oreven HPLC.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide beingassayed always be provided in their most purified state. Indeed, it iscontemplated that less substantially purified products will have utilityin certain embodiments. Partial purification may be accomplished byusing fewer purification steps in combination, or by utilizing differentforms of the same general purification scheme. For example, it isappreciated that a cation-exchange column chromatography performedutilizing an HPLC apparatus will generally result in a greater “-fold”purification than the same technique utilizing a low pressurechromatography system. Methods exhibiting a lower degree of relativepurification may have advantages in total recovery of protein product,or in maintaining the activity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

A particular type of affinity chromatography useful in the purificationof carbohydrate containing compounds is lectin affinity chromatography.Lectins are a class of substances that bind to a variety ofpolysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclams and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

V. Methods for Plant Transformation

Suitable methods for plant transformation for use with the currentinvention are believed to include virtually any method by which DNA canbe introduced into a cell, such as by direct delivery of DNA such as byPEG-mediated transformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), byelectroporation (U.S. Pat. No. 5,384,253, specifically incorporatedherein by reference in its entirety), by agitation with silicon carbidefibers (Kaeppler et al., 1990; U.S. Pat. No. 5,302,523, specificallyincorporated herein by reference in its entirety; and U.S. Pat. No. 105,464,765, specifically incorporated herein by reference in itsentirety), by Agrobacterium-mediated transformation (U.S. Pat. No.5,591,616 and U.S. Pat. No. 5,563,055; both specifically incorporatedherein by reference) and by acceleration of DNA coated particles (U.S.Pat. No. 5,550,318; U.S. Pat. No. 5,538,877; and U.S. Pat. No.5,538,880; each specifically incorporated herein by reference in itsentirety), etc. Through the application of techniques such as these,maize cells as well as those of virtually any other plant species may bestably transformed, and these cells developed into transgenic plants. Incertain embodiments, acceleration methods are preferred and include, forexample, microprojectile bombardment and the like.

(i) Electroporation

Where one wishes to introduce DNA by means of electroporation, it iscontemplated that the method of Krzyzek et al. (U.S. Pat. No. 5,384,253,incorporated herein by reference in its entirety) will be particularlyadvantageous. In this method, certain cell wall-degrading enzymes, suchas pectin-degrading enzymes, are employed to render the target recipientcells more susceptible to transformation by electroporation thanuntreated cells. Alternatively, recipient cells are made moresusceptible to transformation by mechanical wounding.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wounding in acontrolled manner. Examples of some species which have been transformedby electroporation of intact cells include maize (U.S. Pat. No.5,384,253; Rhodes et al., 1995; D'Halluin et al., 1992), wheat (Zhou etal., 1993), tomato (Hou and Lin, 1996), soybean (Christou et al., 1987)and tobacco (Lee et al., 1989).

One also may employ protoplasts for electroporation transformation ofplants (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon-derivedprotoplasts is described by Dhir and Widholm in Intl. Patent Appl. Publ.No. WO 9217598 (specifically incorporated herein by reference). Otherexamples of species for which protoplast transformation has beendescribed include barley (Lazerri, 1995), sorghum (Battraw et al.,1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) andtomato (Tsukada, 1989).

(ii) Microprojectile Bombardment

A preferred method for delivering transforming DNA segments to plantcells in accordance with the invention is microprojectile bombardment(U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No.5,610,042; and PCT Application WO 94/09699; each of which isspecifically incorporated herein by reference in its entirety). In thismethod, particles may be coated with nucleic acids and delivered intocells by a propelling force. Exemplary particles include those comprisedof tungsten, platinum, and preferably, gold. It is contemplated that insome instances DNA precipitation onto metal particles would not benecessary for DNA delivery to a recipient cell using microprojectilebombardment. However, it is contemplated that particles may contain DNArather than be coated with DNA. Hence, it is proposed that DNA-coatedparticles may increase the level of DNA delivery via particlebombardment but are not, in and of themselves, necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a filter surfacecovered with monocot plant cells cultured in suspension. The screendisperses the particles so that they are not delivered to the recipientcells in large aggregates. It is believed that a screen interveningbetween the projectile apparatus and the cells to be bombarded reducesthe size of projectiles aggregate and may contribute to a higherfrequency of transformation by reducing the damage inflicted on therecipient cells by projectiles that are too large.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. Examples of species forwhich have been transformed by microprojectile bombardment includemonocot species such as maize (PCT Application WO 95/06128), barley(Ritala et al., 1994; Hensgens et al., 1993), wheat (U.S. Pat. No.5,563,055, specifically incorporated herein by reference in itsentirety), rice (Hensgens et al., 1993), oat (Torbet et al., 1995;Torbet et al., 1998), rye (Hensgens et al., 1993), sugarcane (Bower etal., 1992), and sorghum (Casa et al., 1993; Hagio et al., 1991); as wellas a number of dicots including tobacco (Tomes et al., 1990; Buising andBenbow, 1994), soybean (U.S. Pat. No. 5,322,783, specificallyincorporated herein by reference in its entirety), sunflower (Knittel etal. 1994), peanut (Singsit et al., 1997), cotton (McCabe and Martinell,1993), tomato (VanEck et al. 1995), and legumes in general (U.S. Pat.No. 5,563,055, specifically incorporated herein by reference in itsentirety).

(iii) Agrobacterium-mediated Transformation

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described by Fraley etal., (1985), Rogers et al., (1987) and U.S. Pat. No. 5,563,055,specifically incorporated herein by reference in its entirety.

Agrobacterium-mediated transformation is most efficient indicotyledonous plants and is the preferable method for transformation ofdicots, including Arabidopsis, tobacco, tomato, and potato. Indeed,while Agrobacterium-mediated transformation has been routinely used withdicotyledonous plants for a number of years, it has only recently becomeapplicable to monocotyledonous plants. Advances inAgrobacterium-mediated transformation techniques have now made thetechnique applicable to nearly all monocotyledonous plants. For example,Agrobacterium-mediated transformation techniques have now been appliedto rice (Hiei et al., 1997; Zhang et al., 1997; U.S. Pat No. 5,591,616,specifically incorporated herein by reference in its entirety), wheat(McCormac et al., 1998), barley (Tingay et al., 1997; McCormac et al.,1998), and maize (Ishidia et al., 1996).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described(Rogers et al., 1987) have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Inaddition, Agrobacterium containing both armed and disarmed Ti genes canbe used for the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

(iv) Other Transformation Methods

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g,Potrykus et al., 1985; Lorz et al., 1985; Omirulleh et al., 1993; Frommet al., 1986; Uchimiya et al., 1986; Callis et al., 1987; Marcotte etal., 1988).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastshave been described (Fujimara et al., 1985; Toriyama et al., 1986;Yamada et al, 1986; Abdullah et al., 1986; Omirulleh et al., 1993 andU.S. Pat. No. 5,508,184; each specifically incorporated herein byreference in its entirety). Examples of the use of direct uptaketransformation of cereal protoplasts include transformation of rice(Ghosh-Biswas et al., 1994), sorghum (Battraw and Hall, 1991), barley(Lazerri, 1995), oat (Zheng and Edwards, 1990) and maize (Omirulleh etal., 1993).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, 1989). Also,silicon carbide fiber-mediated transformation may be used with orwithout protoplasting (Kaeppler, 1990; Kaeppler et al., 1992; U.S. Pat.No. 5,563,055, specifically incorporated herein by reference in itsentirety). Transformation with this technique is accomplished byagitating silicon carbide fibers together with cells in a DNA solution.DNA passively enters as the cell are punctured. This technique has beenused successfully with, for example, the monocot cereals maize (PCTApplication WO 95/06128, specifically incorporated herein by referencein its entirety; Thompson, 1995) and rice (Nagatani, 1997).

VI. Optimization of Microprojectile Bombardment

For microprojectile bombardment transformation in accordance with thecurrent invention, both physical and biological parameters may beoptimized. Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the flight andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, such as the osmotic adjustment of targetcells to help alleviate the trauma associated with bombardment, theorientation of an immature embryo or other target tissue relative to theparticle trajectory, and also the nature of the transforming DNA, suchas linearized DNA or intact supercoiled plasmids. It is believed thatpre-bombardment manipulations are especially important for successfultransformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust various ofthe bombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas DNA concentration, gap distance, flight distance, tissue distance,and helium pressure. It further is contemplated that the grade of heliummay effect transformation efficiency. For example, differences intransformation efficiencies may be witnessed between bombardments usingindustrial grade (99.99% pure) or ultra pure helium (99.999% pure),although it is not currently clear which is more advantageous for use inbombardment. One also may optimize the trauma reduction factors (TRFs)by modifying conditions which influence the physiological state of therecipient cells and which may therefore influence transformation andintegration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation.

(i) Physical Parameters

1. Gap Distance

The variable nest (macro holder) can be adjusted to vary the distancebetween the rupture disk and the macroprojectile, i.e., the gapdistance. This distance can be varied from 0 to 2 cm. The predictedeffects of a shorter gap are an increase of velocity of both the macro-and microprojectiles, an increased shock wave (which leads to tissuesplattering and increased tissue trauma), and deeper penetration ofmicroprojectiles. Longer gap distances would have the opposite effectsbut may increase viability and therefore the total number of recoveredstable transformants.

2. Flight Distance

The fixed nest (contained within the variable nest) can be variedbetween 0.5 and 2.25 cm in predetermined 0.5 cm increments by theplacement of spacer rings to adjust the flight path traversed by themacroprojectile. Short flight paths allow for greater stability of themacroprojectile in flight but reduce the overall velocity of themicroprojectiles. Increased stability in flight increases, for example,the number of centered GUS foci. Greater flight distances (up to somepoint) increase velocity but also increase instability in flight. Basedon observations, it is recommended that bombardments typically be donewith a flight path length of about 1.0 cm to 1.5 cm.

3. Tissue Distance

Placement of tissue within the gun chamber can have significant effectson microprojectile penetration. Increasing the flight path of themicroprojectiles will decrease velocity and trauma associated with theshock wave. A decrease in velocity also will result in shallowerpenetration of the microprojectiles.

4. Helium Pressure

By manipulation of the type and number of rupture disks, pressure can bevaried between 400 and 2000 psi within the gas acceleration tube.Optimum pressure for stable transformation has been determined to bebetween 1000 and 1200 psi.

5. Coating of Microprojectiles.

For microprojectile bombardment, one will attach (i.e. “coat”) DNA tothe microprojectiles such that it is delivered to recipient cells in aform suitable for transformation thereof. In this respect, at least someof the transforming DNA must be available to the target cell fortransformation to occur, while at the same time during delivery the DNAmust be attached to the microprojectile. Therefore, availability of thetransforming DNA from the microprojectile may comprise the physicalreversal of bonds between transforming DNA and the microprojectilefollowing delivery of the microprojectile to the target cell. This neednot be the case, however, as availability to a target cell may occur asa result of breakage of unbound segments of DNA or of other moleculeswhich comprise the physical attachment to the microprojectile.Availability may further occur as a result of breakage of bonds betweenthe transforming DNA and other molecules, which are either directly orindirectly attached to the microprojectile. It further is contemplatedthat transformation of a target cell may occur by way of directrecombination between the transforming DNA and the genomic DNA of therecipient cell. Therefore, as used herein, a “coated” microprojectilewill be one which is capable of being used to transform a target cell,in that the transforming DNA will be delivered to the target cell, yetwill be accessible to the target cell such that transformation mayoccur.

Any technique for coating microprojectiles which allows for delivery oftransforming DNA to the target cells may be used. Methods for coatingmicroprojectiles which have been demonstrated to work well with thecurrent invention have been specifically disclosed herein. DNA may bebound to microprojectile particles using alternative techniques,however. For example, particles may be coated with streptavidin and DNAend labeled with long chain thiol cleavable biotinylated nucleotidechains. The DNA adheres to the particles due to the streptavidin-biotininteraction, but is released in the cell by reduction of the thiollinkage through reducing agents present in the cell.

Alternatively, particles may be prepared by functionalizing the surfaceof a gold oxide particle, providing free amine groups. DNA, having astrong negative charge, binds to the functionalized particles.Furthermore, charged particles may be deposited in controlled arrays onthe surface of mylar flyer disks used in the PDS-1000 Biolistics device,thereby facilitating controlled distribution of particles delivered totarget tissue.

As disclosed above, it further is proposed, that the concentration ofDNA used to coat microprojectiles may influence the recovery oftransformants containing a single copy of the transgene. For example, alower concentration of DNA may not necessarily change the efficiency ofthe transformation, but may instead increase the proportion of singlecopy insertion events. In this regard, approximately 1 ng to 2000 ng oftransforming DNA may be used per each 1.8 mg of startingmicroprojectiles. In other embodiments of the invention, approximately2.5 ng to 1000 ng, 2.5 ng to 750 ng, 2.5 ng to 500 ng, 2.5 ng to 250 ng,2.5 ng to 100 ng, or 2.5 ng to 50 ng of transforming DNA may be used pereach 1.8 mg of starting microprojectiles.

Various other methods also may be used to increase transformationefficiency and/or increase the relative proportion of low-copytransformation events. For example, the inventors contemplateend-modifying transforming DNA with alkaline phosphatase or an agentwhich will blunt DNA ends prior to transformation. Still further, aninert carrier DNA may be included with the transforming DNA, therebylowering the effective transforming DNA concentration without loweringthe overall amount of DNA used. These techniques are further describedin U.S. patent application Ser. No. 08/995,451, filed Dec. 22, 1997, thedisclosure of which is specifically incorporated herein by reference inits entirety.

(ii) Biological Parameters

Culturing conditions and other factors can influence the physiologicalstate of the target cells and may have profound effects ontransformation and integration efficiencies. First, the act ofbombardment could stimulate the production of ethylene which could leadto senescence of the tissue. The addition of antiethylene compoundscould increase transformation efficiencies. Second, it is proposed thatcertain points in the cell cycle may be more appropriate for integrationof introduced DNA. Hence synchronization of cell cultures may enhancethe frequency of production of transformants. For example,synchronization may be achieved using cold treatment, amino acidstarvation, or other cell cycle-arresting agents. Third, the degree oftissue hydration also may contribute to the amount of trauma associatedwith bombardment as well as the ability of the microprojectiles topenetrate cell walls.

The position and orientation of an embryo or other target tissuerelative to the particle trajectory also may be important. For example,the PDS-1000 biolistics device does not produce a uniform spread ofparticles over the surface of a target petri dish. The velocity ofparticles in the center of the plate is higher than the particlevelocity at further distances from the center of the petri dish.Therefore, it is advantageous to situate target tissue on the petri dishsuch as to avoid the center of the dish, referred to by some as the“zone of death.” Furthermore, orientation of the target tissue withregard to the trajectory of targets also can be important. It iscontemplated that it is desirable to orient the tissue most likely toregenerate a plant toward the particle stream. For example, thescutellum of an immature embryo comprises the cells of greatestembryogenic potential and therefore should be oriented toward theparticle stream.

It also has been reported that slightly plasmolyzed yeast cells allowincreased transformation efficiencies (Armaleo et al., 1990). It washypothesized that the altered osmotic state of the cells helped toreduce trauma associated with the penetration of the microprojectile.Additionally, the growth and cell cycle stage may be important withrespect to transformation.

1. Osmotic Adjustment

It has been suggested that osmotic pre-treatment could potentiallyreduce bombardment associated injury as a result of the decreased turgorpressure of the plasmolyzed cell. In a previous study, the number ofcells transiently expressing GUS increased following subculture intoboth fresh medium and osmotically adjusted medium (PCT Application WO95/06128, specifically incorporated herein by reference in itsentirety). Pretreatment times of 90 minutes showed higher numbers of GUSexpressing foci than shorter times. Cells incubated in 500 mOSM/kgmedium for 90 minutes showed an approximately 3.5 fold increase intransient GUS foci than the control. Preferably, immature embryos areprecultured for 4-5 hours prior to bombardment on culture mediumcontaining 12% sucrose. A second culture on 12% sucrose is performed for16-24 hours following bombardment. Alternatively, type II cells arepretreated on 0.2M mannitol for 3-4 hours prior to bombardment. It iscontemplated that pretreatment of cells with other osmotically activesolutes for a period of 1-6 hours also may be desirable.

2. Plasmid Configuration

In some instances, it will be desirable to deliver DNA to maize cellsthat does not contain DNA sequences necessary for maintenance of theplasmid vector in the bacterial host, e.g., E. coli, such as antibioticresistance genes, including but not limited to ampicillin, kanamycin,and tetracycline resistance, and prokaryotic origins of DNA replication.In such case, a DNA fragment containing the transforming DNA may bepurified prior to transformation. An exemplary method of purification isgel electrophoresis on a 1.2% low melting temperature agarose gel,followed by recovery from the agarose gel by melting gel slices in a6-10 fold excess of Tris-EDTA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA,70° C.-72° C.); frozen and thawed (37° C.); and the agarose pelleted bycentrifugation. A Qiagen Q-100 column then may be used for purificationof DNA. For efficient recovery of DNA, the flow rate of the column maybe adjusted to 40 ml/hr.

Isolated DNA fragments can be recovered from agarose gels using avariety of electroelution techniques, enzyme digestion of the agarose,or binding of DNA to glass beads (e.g., Gene Clean). In addition, HPLCand/or use of magnetic particles may be used to isolate DNA fragments.As an alternative to isolation of DNA fragments, a plasmid vector can bedigested with a restriction enzyme and this DNA delivered to maize cellswithout prior purification of the expression cassette fragment.

VII. Recipient Cells for Transformation

Tissue culture requires media and controlled environments. “Media”refers to the numerous nutrient mixtures that are used to grow cells invitro, that is, outside of the intact living organism. The mediumusually is a suspension of various categories of ingredients (salts,amino acids, growth regulators, sugars, buffers) that are required forgrowth of most cell types. However, each specific cell type requires aspecific range of ingredient proportions for growth, and an even morespecific range of formulas for optimum growth. Rate of cell growth alsowill vary among cultures initiated with the array of media that permitgrowth of that cell type.

Nutrient media is prepared as a liquid, but this may be solidified byadding the liquid to materials capable of providing a solid support.Agar is most commonly used for this purpose. Bactoagar, Hazelton agar,Gelrite, and Gelgro are specific types of solid support that aresuitable for growth of plant cells in tissue culture.

Some cell types will grow and divide either in liquid suspension or onsolid media. As disclosed herein, plant cells will grow in suspension oron solid medium, but regeneration of plants from suspension culturestypically requires transfer from liquid to solid media at some point indevelopment. The type and extent of differentiation of cells in culturewill be affected not only by the type of media used and by theenvironment, for example, pH, but also by whether media is solid orliquid. Table 7 illustrates the composition of various media useful forcreation of recipient cells and for plant regeneration.

Recipient cell targets include, but are not limited to, meristem cells,Type I, Type II, and Type III callus, immature embryos and gametic cellssuch as microspores, pollen, sperm and egg cells. It is contemplatedthat any cell from which a fertile plant may be regenerated is useful asa recipient cell. Type I, Type II, and Type III callus may be initiatedfrom tissue sources including, but not limited to, immature embryos,seedling apical meristems, microspores and the like. Those cells whichare capable of proliferating as callus also are recipient cells forgenetic transformation. The present invention provides techniques fortransforming immature embryos and subsequent regeneration of fertiletransgenic plants. Transformation of immature embryos obviates the needfor long term development of recipient cell cultures. Pollen, as well asits precursor cells, microspores, may be capable of functioning asrecipient cells for genetic transformation, or as vectors to carryforeign DNA for incorporation during fertilization Direct pollentransformation would obviate the need for cell culture. Meristematiccells (i e., plant cells capable of continual cell division andcharacterized by an undifferentiated cytological appearance, normallyfound at growing points or tissues in plants such as root tips, stemapices, lateral buds, etc.) may represent another type of recipientplant cell. Because of their undifferentiated growth and capacity fororgan differentiation and totipotency, a single transformed meristematiccell could be recovered as a whole transformed plant. In fact, it isproposed that embryogenic suspension cultures may be an in vitromeristematic cell system, retaining an ability for continued celldivision in an undifferentiated state, controlled by the mediaenvironment.

Cultured plant cells that can serve as recipient cells for transformingwith desired DNA segments may be any plant cells including corn cells,and more specifically, cells from Zea mays L. Somatic cells are ofvarious types. Embryogenic cells are one example of somatic cells whichmay be induced to regenerate a plant through embryo formation.Non-embryogenic cells are those which typically will not respond in sucha fashion. An example of non-embryogenic cells are certain Black MexicanSweet (BMS) corn cells.

The development of embryogenic maize calli and suspension culturesuseful in the context of the present invention, e.g., as recipient cellsfor transformation, has been described in U.S. Pat. No. 5,134,074; andU.S. Pat. No. 5,489,520; each of which is incorporated herein byreference in its entirety.

Certain techniques may be used that enrich recipient cells within a cellpopulation. For example, Type II callus development, followed by manualselection and culture of friable, embryogenic tissue, generally resultsin an enrichment of recipient cells for use in, microprojectiletransformation. Suspension culturing, particularly using the mediadisclosed herein, may improve the ratio of recipient to non-recipientcells in any given population. Manual selection techniques which can beemployed to select recipient cells may include, e.g., assessing cellmorphology and differentiation, or may use various physical orbiological means. Cryopreservation also is a possible method ofselecting for recipient cells.

Manual selection of recipient cells, e.g., by selecting embryogeniccells from the surface of a Type II callus, is one means that may beused in an attempt to enrich for recipient cells prior to culturing(whether cultured on solid media or in suspension). The preferred cellsmay be those located at the surface of a cell cluster, and may furtherbe identifiable by their lack of differentiation, their size and densecytoplasm. The preferred cells will generally be those cells which areless differentiated, or not yet committed to differentiation. Thus, onemay wish to identify and select those cells which are cytoplasmicallydense, relatively unvacuolated with a high nucleus to cytoplasm ratio(e.g., determined by cytological observations), small in size (e.g.,10-20 μm), and capable of sustained divisions and somatic proembryoformation.

It is proposed that other means for identifying such cells also may beemployed. For example, through the use of dyes, such as Evan's blue,which are excluded by cells with relatively non-permeable membranes,such as embryogenic cells, and taken up by relatively differentiatedcells such as root-like cells and snake cells (so-called due to theirsnake-like appearance).

Other possible means of identifying recipient cells include the use ofisozyme markers of embryogenic cells, such as glutamate dehydrogenase,which can be detected by cytochemical stains (Fransz et al., 1989).However, it is cautioned that the use of isozyme markers includingglutamate dehydrogenase may lead to some degree of false positives fromnon-embryogenic cells such as rooty cells which nonetheless have arelatively high metabolic activity.

(i) Culturing Cells to be Recipients for Transformation

The ability to prepare and cryopreserve cultures of plant cells isimportant to certain aspects of the present invention, in that itprovides a means for reproducibly and successfully preparing cells fortransformation. A variety of different types of media have beenpreviously developed and may be employed in carrying out various aspectsof the invention. The following table, Table 7, sets forth thecomposition of the media preferred by the inventor for carrying outthese aspects of the invention.

TABLE 7 Tissue Culture Media Which are Used for Type II CallusDevelopment, Development of Suspension Cultures and Regeneration ofPlant Cells (Particularly Maize Cells) OTHER BASAL COMPONENTS** MEDIANO. MEDIUM SUCROSE pH (Amount/L)   7 MS* 2% 6.0 .25 mg thiamine .5 mgBAP .5 mg NAA Bactoagar  10 MS 2% 6.0 .25 mg thiamine 1 mg BAP 1 mg2,4-D 400 mg L-proline Bactoagar  19 MS 2% 6.0 .25 mg thiamine .25 mgBAP .25 mg NAA Bactoagar  20 MS 3% 6.0 .25 mg thiamine 1 mg BAP 1 mg NAABactoagar  52 MS 2% 6.0 .25 mg thiamine 1 mg 2,4-D 10⁻⁷M ABA BACTOAGAR 101 MS 3% 6.0 MS vitamins 100 mg myo-inositol Bactoagar  105 MS — 3.50.04 mg NAA 3 mg BAP 1 mg thiamine.HCl 0.5 mg niacin 0.91 mgL-asparagine monohydrate 100 mg myo-inositol 100 mg casein hydrolysate1.4 g L-proline 20 g sorbitol 2.0 g Gelgro  110 MS 6% 5.8 1 mgthiamine.HCl 1 mg niacin 3.6 g Gelgro  142 MS 6% 6.0 MS vitamins 5 mgBAP 0.186 mg NAA 0.175 mg IAA 0.403 mg 2IP Bactoagar  157 MS 6% 6.0 MSvitamins 100 mg myo-inositol Bactoagar  163 MS 3% 6.0 MS vitamins 3.3 mgdicamba 100 mg myo-inositol Bactoagar  171 MS 3% 6.0 MS vitamins .25 mg2,4-D 10 mg BAP 100 mg myo-inositol Bactoagar  173 MS 6% 6.0 MS vitamins5 mg BAP .186 mg NAA .175 mg IAA .403 mg 2IP 10⁻⁷M ABA 200 mgmyo-inositol Bactoagar  177 MS 3% 6.0 MS vitamins .25 mg 2,4-D 10 mg BAP10⁻⁷M ABA 100 mg myo-inositol Bactoagar  185 MS — 5.8 3 mg BAP .04 mgNAA RT vitamins 1.65 mg thiamine 1.38 g L-proline 20 g sorbitolBactoagar  189 MS — 5.8 3 mg BAP .04 mg NAA .5 mg niacin 800 mgL-asparagine 100 mg casamino acids 20 g sorbitol 1.4 g L-proline 100 mgmyo-inositol Gelgro  201 N6 2% 5.8 N6 vitamins 2 mg L-glycine 1 mg 2,4-D100 mg casein hydrolysate 2.9 g L-proline Gelgro  205 N6 2% 5.8 N6vitamins 2 mg L-glycine .5 mg 2,4-D 100 mg casein hydrolysate 2.9 gL-proline Gelgro  209 N6 6% 5.8 N6 vitamins 2 mg L-glycine 100 mg caseinhydrolysate 0.69 g L-proline Bactoagar  210 N6 3% 5.5 N6 vitamins 2 mg2,4-D 250 mg Ca pantothenate 100 mg myo-inositol 790 mg L-asparagine 100mg casein hydrolysate 1.4 g L-proline Hazelton agar**** 2 mg L-glycine 211 N6 2% 5.8 1 mg 2,4-D 0.5 mg niacin 1.0 mg thiamine 0.91 gL-asparagine 100 mg myo-inositol 0.5 g MES 100 mg/L casein hydrolysate1.6 g MgCl₂-6H₂O 0.69 g L-proline 2 g Gelgro  212 N6 3% 5.5 N6 vitamins2 mg L-glycine 2 mg 2,4-D 250 mg Ca pantothenate 100 mg myo-inositol 100mg casein hydrolysate 1.4 g L-proline Hazelton agar****  227 N6 2% 5.8N6 vitamins 2 mg L-glycine 13.2 mg dicamba 100 mg casein hydrolysate 2.9g L-proline Gelgro  273 N6 2% 5.8 N6 vitamins (also, 201V, 2 mgL-glycine 236S, 201D, 1 mg 2,4-D 2071, 2366, 16.9 mg AgNO₃ 201SV, 2377,100 mg casein and 201BV) hydrolysate 2.9 g L-proline  279 N6 2% 5.8 3.3mg dicamba 1 mg thiamine .5 mg niacin 800 mg L-asparagine 100 mg caseinhydrolysate 100 mg myoinositol 1.4 g L-proline Gelgro****  288 N6 3% 3.3mg dicamba 1 mg thiamine .5 mg niacin .8 g L-asparagine 100 mgmyo-inosital 1.4 g L-proline 100 mg casein hydrolysate 16.9 mg AgNO₃Gelgro  401 MS 3% 6.0 3.73 mg Na₂EDTA .25 mg thiamine 1 mg 2,4-D 2 mgNAA 200 mg casein hydrolysate 500 mg K₂SO₄ 400 mg KH₂PO₄ 100 mgmyo-inositol  402 MS 3% 6.0 3.73 mg Na₂EDTA .25 mg thiamine 1 mg 2,4-D200 mg casein hydrolysate 2.9 g L-proline 500 mg K₂SO₄ 400 mg KH₂PO₄ 100mg myo-inositol  409 MS 3% 6.0 3.73 mg Na₂EDTA .25 mg thiamine 9.9 mgdicamba 200 mg casein hydrolysate 2.9 g L-proline 500 mg K₂SO₄ 400 mgKH₂PO₄ 100 mg myo-inositol  501 Clark's 2% 5.7 Medium***  607 1/2 × MS3% 5.8 1 mg thiamine 1 mg niacin Gelrite  615 MS 3% 6.0 MS vitamins 6 mgBAP 100 mg myo-inositol Bactoagar  617 1/2 × MS   1.5% 6.0 MS vitamins50 mg myo-inositol Bactoagar  708 N6 2% 5.8 N6 vitamins 2 mg L-glycine1.5 mg 2,4-D 200 mg casein hydrolysate 0.69 g L-proline Gelrite  721 N62% 5.8 3.3 mg dicamba 1 mg thiamine .5 mg niacin 800 mg L-asparagine 100mg myo-inositol 100 mg casein hydrolysate 1.4 g L-proline 54.65 gmannitol Gelgro  726 N6 3% 5.8 3.3 mg dicamba .5 mg niacin 1 mg thiamine800 mg L-asparagine 100 mg myo-inositol 100 mg casein hydrolysate 1.4 gL-proline  727 N6 3% 5.8 N6 vitamins 2 mg L-glycine 9.9 mg dicamba 100mg casein hydrolysate 2.9 g L-proline Gelgro  728 N6 3% 5.8 N6 vitamins2 mg L-glycine 9.9 mg dicamba 16.9 mg AgNO₃ 100 mg casein hydrolysate2.9 g L-proline Gelgro  734 N6 2% 5.8 N6 vitamins 2 mg L-glycine 1.5 mg2,4-D 14 g Fe sequestreene (replaces Fe-EDTA) 200 mg casein hydrolyste0.69 g L-proline Gelrite  735 N6 2% 5.8 1 mg 2,4-D .5 mg niacin .91 gL-asparagine 100 mg myo-inositol 1 mg thiamine .5 g MES .75 g MgCl₂ 100mg casein hydrolysate 0.69 g L-proline Gelgro 2004 N6 3% 5.8 1 mgthiamine 0.5 mg niacin 3.3 mg dicamba 17 mg AgNO₃ 1.4 g L-proline 0.8 gL-asparagine 100 mg casein hydrolysate 100 mg myo-inositol Gelrite 2008N6 3% 5.8 1 mg thiamine 0.5 mg niacin 3.3 mg dicamba 1.4 g L-proline 0.8g L-asparagine Gelrite *Basic MS medium discribed in Murashige and Skoog(1962). This medium is typically modified by decreasing the NH₄NO₃ from1.64 g/l to 1.55 g/l, and omitting the pyridoxine HCl, nicotinic acid,myo-inositol and glycine. **NAA = Napthol Acetic Acid IAA = IndoleAcetic Acid 2-IP = 2, isopentyl adenine 2,4-D =2,4-Dichlorophenoxyacetic Acid BAP = 6-Benzyl aminopurine ABA = abscisicacid ***Basic medium described in Clark (1982) ****These media may bemade with or without solidifying agent.

A number of exemplary maize cultures which may be used fortransformation have been developed and are disclosed in U.S. patentappl. Ser. No. 08/113,561, filed Aug. 25, 1993, which is specificallyincorporated herein by reference.

(ii) Media

In certain embodiments of the current invention, recipient cells may beselected following growth in culture. Where employed, cultured cells maybe grown either on solid supports or in the form of liquid suspensions.In either instance, nutrients may be provided to the cells in the formof media, and environmental conditions controlled. There are many typesof tissue culture media comprised of various amino acids, salts, sugars,growth regulators and vitamins. Most of the media employed in thepractice of the invention will have some similar components (see Table7), but may differ in the composition and proportions of theiringredients depending on the particular application envisioned. Forexample, various cell types usually grow in more than one type of media,but will exhibit different growth rates and different morphologies,depending on the growth media. In some media, cells survive but do notdivide.

Various types of media suitable for culture of plant cells previouslyhave been described. Examples of these media include, but are notlimited to, the N6 medium described by Chu et al. (1975) and MS media(Murashige and Skoog, 1962). It has been discovered that media such asMS which have a high ammonia/nitrate ratio are counterproductive to thegeneration of recipient cells in that they promote loss of morphogeniccapacity. N6 media, on the other hand, has a somewhat lowerammonia/nitrate ratio, and is contemplated to promote the generation ofrecipient cells by maintaining cells in a proembryonic state capable ofsustained divisions.

(iii) Maintenance

The method of maintenance of cell cultures may contribute to theirutility as sources of recipient cells for transformation. Manualselection of cells for transfer to fresh culture medium, frequency oftransfer to fresh culture medium, composition of culture medium, andenvironmental factors including, but not limited to, light quality andquantity and temperature are all important factors in maintaining callusand/or suspension cultures that are useful as sources of recipientcells. It is contemplated that alternating callus between differentculture conditions may be beneficial in enriching for recipient cellswithin a culture. For example, it is proposed that cells may be culturedin suspension culture, but transferred to solid medium at regularintervals. After a period of growth on solid medium cells can bemanually selected for return to liquid culture medium. It is proposedthat by repeating this sequence of transfers to fresh culture medium itis possible to enrich for recipient cells. It also is contemplated thatpassing cell cultures through a 1.9 mm sieve is useful in maintainingthe friability of a callus or suspension culture and may be beneficialin enriching for transformable cells.

(iv) Cryopreservation Methods

Cryopreservation is important because it allows one to maintain andpreserve a known transformable cell culture for future use, whileeliminating the cumulative detrimental effects associated with extendedculture periods.

Cell suspensions and callus were cryopreserved using modifications ofmethods previously reported (Finkle, 1985; Withers & King, 1979). Thecryopreservation protocol comprised adding a pre-cooled (0° C.)concentrated cryoprotectant mixture stepwise over a period of one to twohours to pre-cooled (0° C.) cells. The mixture was maintained at 0° C.throughout this period. The volume of added cryoprotectant was equal tothe initial volume of the cell suspension (1:1 addition), and the finalconcentration of cryoprotectant additives was 10% dimethyl sulfoxide,10% polyethylene glycol (6000 MW), 0.23M proline and 0.23M glucose. Themixture was allowed to equilibrate at 0° C. for 30 minutes, during whichtime the cell suspension/ cryoprotectant mixture was divided into 1.5 mlaliquot (0.5 ml packed cell volume) in 2 ml polyethylene cryo-vials. Thetubes were cooled at 0.5° C./minute to −8° C. and held at thistemperature for ice nucleation.

Once extracellular ice formation had been visually confirmed, the tubeswere cooled at 0.5° C./minute from −8° C. to −35° C. They were held atthis temperature for 45 minutes (to insure uniform freeze-induceddehydration throughout the cell clusters). At this point, the cells hadlost the majority of their osmotic volume (i.e., there is little freewater left in the cells), and they could be safely plunged into liquidnitrogen for storage. The paucity of free water remaining in the cellsin conjunction with the rapid cooling rates from −35° C. to −196° C.prevented large organized ice crystals from forming in the cells. Thecells are stored in liquid nitrogen, which effectively immobilizes thecells and slows metabolic processes to the point where long-term storageshould not be detrimental.

Thawing of the extracellular solution was accomplished by removing thecryo-tube from liquid nitrogen and swirling it in sterile 42° C. waterfor approximately 2 minutes. The tube was removed from the heatimmediately after the last ice crystals had melted to prevent heatingthe tissue. The cell suspension (still in the cryoprotectant mixture)was pipetted onto a filter, resting on a layer of BMS cells (the feederlayer which provided a nurse effect during recovery). The cryoprotectantsolution is removed by pipetting. Culture medium comprised a callusproliferation medium with increased osmotic strength. Dilution of thecryoprotectant occurred slowly as the solutes diffused away through thefilter and nutrients diffused upward to the recovering cells. Oncesubsequent growth of the thawed cells was noted, the growing tissue wastransferred to fresh culture medium. If initiation of a suspensionculture was desired, the cell clusters were transferred back into liquidsuspension medium as soon as sufficient cell mass had been regained(usually within 1 to 2 weeks). Alternatively, cells were cultured onsolid callus proliferation medium. After the culture was reestablishedin liquid (within 1 to 2 additional weeks), it was used fortransformation experiments. When desired, previously cryopreservedcultures may be frozen again for storage.

VIII. Production and Characterization of Stably Transformed Plants

After effecting delivery of exogenous DNA to recipient cells, the nextsteps generally concern identifying the transformed cells for furtherculturing and plant regeneration. As mentioned herein, in order toimprove the ability to identify transformants, one may desire to employa selectable or screenable marker gene as, or in addition to, theexpressible gene of interest. In this case, one would then generallyassay the potentially transformed cell population by exposing the cellsto a selective agent or agents, or one would screen the cells for thedesired marker gene trait.

(i) Selection

It is believed that DNA is introduced into only a small percentage oftarget cells in any one experiment. In order to provide an efficientsystem for identification of those cells receiving DNA and integratingit into their genomes one may employ a means for selecting those cellsthat are stably transformed. One exemplary embodiment of such a methodis to introduce into the host cell, a marker gene which confersresistance to some normally inhibitory agent, such as an antibiotic orherbicide. Examples of antibiotics which may be used include theaminoglycoside antibiotics neomycin, kanamycin and paromomycin, or theantibiotic hygromycin. Resistance to the aminoglycoside antibiotics isconferred by arninoglycoside phosphostransferase enzymes such asneomycin phosphotransferase II (NPT II) or NPT I, whereas resistance tohygromycin is conferred by hygromycin phosphotransferase.

Potentially transformed cells then are exposed to the selective agent.In the population of surviving cells will be those cells where,generally, the resistance-conferring gene has been integrated andexpressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of the exogenous DNA. Usingthe techniques disclosed herein, greater than 40% of bombarded embryosmay yield transformants.

One herbicide which constitutes a desirable selection agent is the broadspectrum herbicide bialaphos. Bialaphos is a tripeptide antibioticproduced by Streptomyces hygroscopicus and is composed ofphosphinothricin (PPT), an analogue of L-glutamic acid, and twoL-alanine residues. Upon removal of the L-alanine residues byintracellular peptidases, the PPT is released and is a potent inhibitorof glutamine synthetase (GS), a pivotal enzyme involved in ammoniaassimilation and nitrogen metabolism (Ogawa et al., 1973). SyntheticPPT, the active ingredient in the herbicide Liberty™ also is effectiveas a selection agent. Inhibition of GS in plants by PPT causes the rapidaccumulation of ammonia and death of the plant cells.

The organism producing bialaphos and other species of the genusStreptomyces also synthesizes an enzyme phosphinothricin acetyltransferase (PAT) which is encoded by the bar gene in Streptomyceshygroscopicus and the pat gene in Streptomyces viridochromogenes. Theuse of the herbicide resistance gene encoding phosphinothricin acetyltransferase (PAT) is referred to in DE 3642 829 A, wherein the gene isisolated from Streptomyces viridochromogenes. In the bacterial sourceorganism, this enzyme acetylates the free amino group of PPT preventingauto-toxicity (Thompson et al., 1987). The bar gene has been cloned(Murakami et al., 1986; Thompson et al., 1987) and expressed intransgenic tobacco, tomato, potato (De Block et al., 1987) Brassica (DeBlock et al., 1989) and maize (U.S. Pat. No. 5,550,318). In previousreports, some transgenic plants which expressed the resistance gene werecompletely resistant to commercial formulations of PPT and bialaphos ingreenhouses.

Another example of a herbicide which is useful for selection oftransformed cell lines in the practice of the invention is the broadspectrum herbicide glyphosate. Glyphosate inhibits the action of theenzyme EPSPS which is active in the aromatic amino acid biosyntheticpathway. Inhibition of this enzyme leads to starvation for the aminoacids phenylalanine, tyrosine, and tryptophan and secondary metabolitesderived thereof. U.S. Pat. No. 4,535,060 describes the isolation ofEPSPS mutations which confer glyphosate resistance on the Salmonellatyphimurium gene for EPSPS, aroA. The EPSPS gene was cloned from Zeamays and mutations similar to those found in a glyphosate resistant aroAgene were introduced in vitro. Mutant genes encoding glyphosateresistant EPSPS enzymes are described in, for example, InternationalPatent WO 97/4103. The best characterized mutant EPSPS gene conferringglyphosate resistance comprises amino acid changes at residues 102 and106, although it is anticipated that other mutations will also be useful(PCT/WO97/4103).

To use the bar-bialaphos or the EPSPS-glyphosate selective system,bombarded tissue is cultured for 0-28 days on nonselective medium andsubsequently transferred to medium containing from 1-3 mg/l bialaphos or1-3 mM glyphosate as appropriate. While ranges of 1-3 mg/l bialaphos or1-3 mM glyphosate will typically be preferred, it is proposed thatranges of 0.1-50 mg/l bialaphos or 0.1-50 mM glyphosate will findutility in the practice of the invention. Tissue can be placed on anyporous, inert, solid or semi-solid support for bombardment, includingbut not limited to filters and solid culture medium. Bialaphos andglyphosate are provided as examples of agents suitable for selection oftransformants, but the technique of this invention is not limited tothem.

It further is contemplated that the herbicide DALAPON,2,2-dichloropropionic acid, may be useful for identification oftransformed cells. The enzyme 2,2-dichloropropionic acid dehalogenase(deh) inactivates the herbicidal activity of 2,2-dichloropropionic acidand therefore confers herbicidal resistance on cells or plantsexpressing a gene encoding the dehalogenase enzyme (Buchanan-Wollastonet al., 1992; U.S. patent appl. Ser. No. 08/113,561, filed Aug. 25,1993; U.S. Pat. No. 5,508,468; and U.S. Pat. No. 5,508,468; each of thedisclosures of which is specifically incorporated herein by reference inits entirety).

Alternatively, a gene encoding anthranilate synthase, which confersresistance to certain amino acid analogs, e.g., 5-methyltryptophan or6-methyl anthranilate, may be useful as a selectable marker gene. Theuse of an anthranilate synthase gene as a selectable marker wasdescribed in U.S. Pat. No. 5,508,468; and U.S. patent application Ser.No. 08/604,789.

An example of a screenable marker trait is the red pigment producedunder the control of the R-locus in maize. This pigment may be detectedby culturing cells on a solid support containing nutrient media capableof supporting growth at this stage and selecting cells from colonies(visible aggregates of cells) that are pigmented. These cells may becultured further, either in suspension or on solid media. The R-locus isuseful for selection of transformants from bombarded immature embryos.In a similar fashion, the introduction of the Cl and B genes will resultin pigmented cells and/or tissues.

The enzyme luciferase may be used as a screenable marker in the contextof the present invention. In the presence of the substrate luciferin,cells expressing luciferase emit light which can be detected onphotographic or x-ray film, in a luminometer (or liquid scintillationcounter), by devices that enhance night vision, or by a highly lightsensitive video camera, such as a photon counting camera. All of theseassays are nondestructive and transformed cells may be cultured furtherfollowing identification. The photon counting camera is especiallyvaluable as it allows one to identify specific cells or groups of cellswhich are expressing luciferase and manipulate those in real time.Another screenable marker which may be used in a similar fashion is thegene coding for green fluorescent protein.

It further is contemplated that combinations of screenable andselectable markers will be useful for identification of transformedcells. In some cell or tissue types a selection agent, such as bialaphosor glyphosate, may either not provide enough killing activity to clearlyrecognize transformed cells or may cause substantial nonselectiveinhibition of transformants and nontransformants alike, thus causing theselection technique to not be effective. It is proposed that selectionwith a growth inhibiting compound, such as bialaphos or glyphosate atconcentrations below those that cause 100% inhibition followed byscreening of growing tissue for expression of a screenable marker genesuch as luciferase would allow one to recover transformants from cell ortissue types that are not amenable to selection alone. It is proposedthat combinations of selection and screening may enable one to identifytransformants in a wider variety of cell and tissue types. This may beefficiently achieved using a gene fusion between a selectable markergene and a screenable marker gene, for example, between an NPTII geneand a GFP gene.

(ii) Regeneration and Seed Production

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. In an exemplary embodiment, MS andN6 media may be modified (see Table 7) by including further substancessuch as growth regulators. A preferred growth regulator for suchpurposes is dicamba or 2,4-D. However, other growth regulators may beemployed, including NAA, NAA+ 2,4-D or perhaps even picloram. Mediaimprovement in these and like ways has been found to facilitate thegrowth of cells at specific developmental stages. Tissue may bemaintained on a basic media with growth regulators until sufficienttissue is available to begin plant regeneration efforts, or followingrepeated rounds of manual selection, until the morphology of the tissueis suitable for regeneration, at least 2 wk, then transferred to mediaconducive to maturation of embryoids. Cultures are transferred every 2wk on this medium. Shoot development will signal the time to transfer tomedium lacking growth regulators.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, will then beallowed to mature into plants. Developing plantlets are transferred tosoiless plant growth mix, and hardened, e.g., in an environmentallycontrolled chamber at about 85% relative humidity, 600 ppm CO₂, and25-250 microeinsteins m⁻² s⁻¹ of light. Plants are preferably maturedeither in a growth chamber or greenhouse. Plants are regenerated fromabout 6 wk to 10 months after a transformant is identified, depending onthe initial tissue. During regeneration, cells are grown on solid mediain tissue culture vessels. Illustrative embodiments of such vessels arepetri dishes and Plant Cons. Regenerating plants are preferably grown atabout 19 to 28° C. After the regenerating plants have reached the stageof shoot and root development, they may be transferred to a greenhousefor further growth and testing.

Note, however, that seeds on transformed plants may occasionally requireembryo rescue due to cessation of seed development and prematuresenescence of plants. To rescue developing embryos, they are excisedfrom surface-disinfected seeds 10-20 days post-pollination and cultured.An embodiment of media used for culture at this stage comprises MSsalts, 2% sucrose, and 5.5 g/l agarose. In embryo rescue, large embryos(defined as greater than 3 mm in length) are germinated directly on anappropriate media. Embryos smaller than that may be cultured for 1 wk onmedia containing the above ingredients along with 10⁻⁵M abscisic acidand then transferred to growth regulator-free medium for germination.

Progeny may be recovered from transformed plants and tested forexpression of the exogenous expressible gene by localized application ofan appropriate substrate to plant parts such as leaves. In the case ofbar transformed plants, it was found that transformed parental plants(R₀) and their progeny of any generation tested exhibited nobialaphos-related necrosis after localized application of the herbicideBasta to leaves, if there was functional PAT activity in the plants asassessed by an in vitro enzymatic assay. All PAT positive progeny testedcontained bar, confirming that the presence of the enzyme and theresistance to bialaphos were associated with the transmission throughthe germline of the marker gene.

(iii) Characterization

To confirm the presence of the exogenous DNA or “transgene(s)” in theregenerating plants, a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays, such as Southernand Northern blotting and PCRT; “biochemical” assays, such as detectingthe presence of a protein product, e.g., by immunological means (ELISAsand Western blots) or by enzymatic function; plant part assays, such asleaf or root assays; and also, by analyzing the phenotype of the wholeregenerated plant.

1. DNA Integration, RNA Expression and Inheritance

Genomic DNA may be isolated from callus cell lines or any plant parts todetermine the presence of the exogenous gene through the use oftechniques well known to those skilled in the art. Note, that intactsequences will not always be present, presumably due to rearrangement ordeletion of sequences in the cell.

The presence of DNA elements introduced through the methods of thisinvention may be determined by polymerase chain reaction (PCR™). Usingthis technique discreet fragments of DNA are amplified and detected bygel electrophoresis. This type of analysis permits one to determinewhether a gene is present in a stable transformant, but does not proveintegration of the introduced gene into the host cell genome It is theexperience of the inventor, however, that DNA has been integrated intothe genome of all transformants that demonstrate the presence of thegene through PCR™ analysis. In addition, it is not possible using PCR™techniques to determine whether transformants have exogenous genesintroduced into different sites in the genome, i.e., whethertransformants are of independent origin. It is contemplated that usingPCR™ techniques it would be possible to clone fragments of the hostgenomic DNA adjacent to an introduced gene.

Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition it is possible through Southernhybridization to demonstrate the presence of introduced genes in highmolecular weight DNA, ie., confirm that the introduced gene has beenintegrated into the host cell genome. The technique of Southernhybridization provides information that is obtained using PCR™, e.g.,the presence of a gene, but also demonstrates integration into thegenome and characterizes each individual transformant.

It is contemplated that using the techniques of dot or slot blothybridization which are modifications of Southern hybridizationtechniques one could obtain the same information that is derived fromPCR™, e.g., the presence of a gene.

Both PCR™ and Southern hybridization techniques can be used todemonstrate transmission of a transgene to progeny. In most instancesthe characteristic Southern hybridization pattern for a giventransformant will segregate in progeny as one or more Mendelian genes(Spencer et al., 1992) indicating stable inheritance of the transgene.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA will only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR™ techniques also may be used for detection andquantitation of RNA produced from introduced genes. In this applicationof PCR™ it is first necessary to reverse transcribe RNA into DNA, usingenzymes such as reverse transcriptase, and then through the use ofconventional PCR™ techniques amplify the DNA. In most instances PCR™techniques, while useful, will not demonstrate integrity of the RNAproduct. Further information about the nature of the RNA product may beobtained by Northern blotting. This technique will demonstrate thepresence of an RNA species and give information about the integrity ofthat RNA. The presence or absence of an RNA species also can bedetermined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and will onlydemonstrate the presence or absence of an RNA species.

2. Gene Expression

While Southern blotting and PCR™ may be used to detect the gene(s) inquestion, they do not provide information as to whether the gene isbeing expressed. Expression may be evaluated by specifically identifyingthe protein products of the introduced genes or evaluating thephenotypic changes brought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

Assay procedures also may be used to identify the expression of proteinsby their functionality, especially the ability of enzymes to catalyzespecific chemical reactions involving specific substrates and products.These reactions may be followed by providing and quantifying the loss ofsubstrates or the generation of products of the reactions by physical orchemical procedures. Examples are as varied as the enzyme to be analyzedand may include assays for PAT enzymatic activity by followingproduction of radiolabeled acetylated phosphinothricin fromphosphinothricin and ¹⁴C-acetyl CoA or for anthranilate synthaseactivity by following loss of fluorescence of anthranilate, to name two.

Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of genesencoding enzymes or storage proteins which change amino acid compositionand may be detected by amino acid analysis, or by enzymes which changestarch quantity which may be analyzed by near infrared reflectancespectrometry. Morphological changes may include greater stature orthicker stalks. Most often changes in response of plants or plant partsto imposed treatments are evaluated under carefully controlledconditions termed bioassays.

IX. Site Specific Integration or Excision of Transgenes

It is specifically contemplated by the inventors that one could employtechniques for the site-specific integration or excision oftransformation constructs prepared in accordance with the instantinvention. An advantage of site-specific integration or excision is thatit can be used to overcome problems associated with conventionaltransformation techniques, in which transformation constructs typicallyrandomly integrate into a host genome in multiple copies. This randominsertion of introduced DNA into the genome of host cells can be lethalif the foreign DNA inserts into an essential gene. In addition, theexpression of a transgene may be influenced by “position effects” causedby the surrounding genomic DNA. Further, because of difficultiesassociated with plants possessing multiple transgene copies, includinggene silencing, recombination and unpredictable inheritance, it istypically desirable to control the copy number of the inserted DNA,often only desiring the insertion of a single copy of the DNA sequence.

Site-specific integration or excision of transgenes or parts oftransgenes can be achieved in plants by means of homologousrecombination (see, for example, U.S. Pat. No. 5,527,695, specificallyincorporated herein by reference in its entirety). Homologousrecombination is a reaction between any pair of DNA sequences having asimilar sequence of nucleotides, where the two sequences interact(recombine) to form a new recombinant DNA species. The frequency ofhomologous recombination increases as the length of the sharednucleotide DNA sequences increases, and is higher with linearizedplasmid molecules than with circularized plasmid molecules. Homologousrecombination can occur between two DNA sequences that are less thanidentical, but the recombination frequency declines as the divergencebetween the two sequences increases.

Introduced DNA sequences can be targeted via homologous recombination bylinking a DNA molecule of interest to sequences sharing homology withendogenous sequences of the host cell. Once the DNA enters the cell, thetwo homologous sequences can interact to insert the introduced DNA atthe site where the homologous genomic DNA sequences were located.Therefore, the choice of homologous sequences contained on theintroduced DNA will determine the site where the introduced DNA isintegrated via homologous recombination. For example, if the DNAsequence of interest is linked to DNA sequences sharing homology to asingle copy gene of a host plant cell, the DNA sequence of interest willbe inserted via homologous recombination at only that single specificsite. However, if the DNA sequence of interest is linked to DNAsequences sharing homology to a multicopy gene of the host eukaryoticcell, then the DNA sequence of interest can be inserted via homologousrecombination at each of the specific sites where a copy of the gene islocated.

DNA can be inserted into the host genome by a homologous recombinationreaction involving either a single reciprocal recombination (resultingin the insertion of the entire length of the introduced DNA) or througha double reciprocal recombination (resulting in the insertion of onlythe DNA located between the two recombination events). For example, ifone wishes to insert a foreign gene into the genomic site where aselected gene is located, the introduced DNA should contain sequenceshomologous to the selected gene. A single homologous recombination eventwould then result in the entire introduced DNA sequence being insertedinto the selected gene. Alternatively, a double recombination event canbe achieved by flanking each end of the DNA sequence of interest (thesequence intended to be inserted into the genome) with DNA sequenceshomologous to the selected gene. A homologous recombination eventinvolving each of the homologous flanking regions will result in theinsertion of the foreign DNA. Thus only those DNA sequences locatedbetween the two regions sharing genomic homology become integrated intothe genome.

Although introduced sequences can be targeted for insertion into aspecific genomic site via homologous recombination, in higher eukaryoteshomologous recombination is a relatively rare event compared to randominsertion events. In plant cells, foreign DNA molecules find homologoussequences in the cell's genome and recombine at a frequency ofapproximately 0.5-4.2×10⁻⁴. Thus any transformed cell that contains anintroduced DNA sequence integrated via homologous recombination willalso likely contain numerous copies of randomly integrated introducedDNA sequences. Therefore, to maintain control over the copy number andthe location of the inserted DNA, these randomly inserted DNA sequencescan be removed. One manner of removing these random insertions is toutilize a site-specific recombinase system. In general, a site specificrecombinase system consists of three elements: two pairs of DNA sequence(the site-specific recombination sequences) and a specific enzyme (thesite-specific recombinase). The site-specific recombinase will catalyzea recombination reaction only between two site-specific recombinationsequences.

A number of different site specific recombinase systems could beemployed in accordance with the instant invention, including, but notlimited to, the Cre/lox system of bacteriophage P1 (U.S. Pat. No.5,658,772, specifically incorporated herein by reference in itsentirety), the FLP/FRT system of yeast (Golic and Lindquist, 1989), theGin recombinase of phage Mu (Maeser et al., 1991), the Pin recombinaseof E. coli (Enomoto et al., 1983), and the R/RS system of the pSR1plasmid (Araki et al., 1992). The bacteriophage P1 Cre/lox and the yeastFLP/FRT systems constitute two particularly useful systems for sitespecific integration or excision of transgenes. In these systems arecombinase (Cre or FLP) will interact specifically with its respectivesite-specific recombination sequence (lox or FRT, respectively) toinvert or excise the intervening sequences. The sequence for each ofthese two systems is relatively short (34 bp for lox and 47 bp for FRT)and therefore, convenient for use with transformation vectors.

The FLP/FRT recombinase system has been demonstrated to functionefficiently in plant cells. Experiments on the performance of theFLP/FRT system in both maize and rice protoplasts indicate that FRT sitestructure, and amount of the FLP protein present, affects excisionactivity. In general, short incomplete FRT sites leads to higheraccumulation of excision products than the complete full-length FRTsites. The systems can catalyze both intra- and intermolecular reactionsin maize protoplasts, indicating its utility for DNA excision as well asintegration reactions. The recombination reaction is reversible and thisreversibility can compromise the efficiency of the reaction in eachdirection. Altering the structure of the site-specific recombinationsequences is one approach to remedying this situation. The site-specificrecombination sequence can be mutated in a manner that the product ofthe recombination reaction is no longer recognized as a substrate forthe reverse reaction, thereby stabilizing the integration or excisionevent.

In the Cre-lox system, discovered in bacteriophage P1, recombinationbetween loxP sites occurs in the presence of the Cre recombinase (see,e.g., U.S. Pat. No. 5,658,772, specifically incorporated herein byreference in its entirety). This system has been utilized to excise agene located between two lox sites which had been introduced into ayeast genome (Sauer, 1987). Cre was expressed from an inducible yeastGALI promoter and this Cre gene was located on an autonomouslyreplicating yeast vector.

Since the lox site is an asymmetrical nucleotide sequence, lox sites onthe same DNA molecule can have the same or opposite orientation withrespect to each other. Recombination between lox sites in the sameorientation results in a deletion of the DNA Segment located between thetwo lox sites and a connection between the resulting ends of theoriginal DNA molecule. The deleted DNA segment forms a circular moleculeof DNA. The original DNA molecule and the resulting circular moleculeeach contain a single lox site. Recombination between lox sites inopposite orientations on the same DNA molecule result in an inversion ofthe nucleotide sequence of the DNA segment located between the two loxsites. In addition, reciprocal exchange of DNA segments proximate to loxsites located on two different DNA molecules can occur. All of theserecombination events are catalyzed by the product of the Cre codingregion.

X. Breeding Plants of the Invention

In addition to direct transformation of a particular genotype with aconstruct prepared according to the current invention, transgenic plantsmay be made by crossing a plant having a construct of the invention to asecond plant lacking the construct. For example, a selected codingregion operably linked to an RS81 promoter can be introduced into aparticular plant variety by crossing, without the need for ever directlytransforming a plant of that given variety. Therefore, the currentinvention not only encompasses a plant directly regenerated from cellswhich have been transformed in accordance with the current invention,but also the progeny of such plants. As used herein the term “progeny”denotes the offspring of any generation of a parent plant prepared inaccordance with the instant invention, wherein the progeny comprises aconstruct prepared in accordance with the invention. “Crossing” a plantto provide a plant line having one or more added transgenes relative toa starting plant line, as disclosed herein, is defined as the techniquesthat result in a transgene of the invention being introduced into aplant line by crossing a starting line with a donor plant line thatcomprises a transgene of the invention. To achieve this one could, forexample, perform the following steps:

(a) plant seeds of the first (starting line) and second (donor plantline that comprises a transgene of the invention) parent plants;

(b) grow the seeds of the first and second parent plants into plantsthat bear flowers;

(c) pollinate the female flower of the first parent plant with thepollen of the second parent plant; and

(d) harvest seeds produced on the parent plant bearing the femaleflower.

Backcrossing is herein defined as the process including the steps of:

(a) crossing a plant of a first genotype containing a desired gene, DNAsequence or element to a plant of a second genotype lacking said desiredgene, DNA sequence or element;

(b) selecting one or more progeny plant containing the desired gene, DNAsequence or element;

(c) crossing the progeny plant to a plant of the second genotype; and

(d) repeating steps (b) and (c) for the purpose of transferring saiddesired gene, DNA sequence or element from a plant of a first genotypeto a plant of a second genotype.

Introgression of a DNA element into a plant genotype is defined as theresult of the process of backcross conversion. A plant genotype intowhich a DNA sequence has been introgressed may be referred to as abackcross converted genotype, line, inbred, or hybrid. Similarly a plantgenotype lacking said desired DNA sequence may be referred to as anunconverted genotype, line, inbred, or hybrid.

XI. Definitions

Genetic Transformation: A process of introducing a DNA sequence orconstruct (e.g., a vector or expression cassette) into a cell orprotoplast in which that exogenous DNA is incorporated into a chromosomeor is capable of autonomous replication.

Exogenous gene: A gene which is not normally present in a given hostgenome in the exogenous gene's present form In this respect, the geneitself may be native to the host genome, however, the exogenous genewill comprise the native gene altered by the addition or deletion of oneor more different regulatory elements.

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a coding DNA molecule such asa structural gene to produce a polypeptide.

Expression cassette: A chimeric DNA molecule which is designed forintroduction into a host genome by genetic transformation. Preferredexpression cassettes will comprise all of the genetic elements necessaryto direct the expression of a selected gene. Expression cassettesprepared in accordance with the instant invention will include an RS81promoter.

Expression vector: A vector comprising at least one expression cassette.

Obtaining: When used in conjunction with a transgenic plant cell ortransgenic plant, obtaining means either transforming a non-transgenicplant cell or plant to create the transgenic plant cell or plant, orplanting transgenic plant seed to produce the transgenic plant cell orplant.

Progeny: Any subsequent generation, including the seeds and plantstherefrom, which is derived from a particular parental plant or set ofparental plants.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provide an expression control element for a structural gene and towhich RNA polymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

R₀ Transgenic Plant: A plant which has been directly transformed with aselected DNA or has been regenerated from a cell or cell cluster whichhas been transformed with a selected DNA.

Regeneration: The process of growing a plant from a plant cell (e.g.,plant protoplast, callus or explant).

Selected DNA: A DNA segment which one desires to introduce into a plantgenome by genetic transformation.

Selected Gene: A gene which one desires to have expressed in atransgenic plant, plant cell or plant part. A selected gene may benative or foreign to a host genome, but where the selected gene ispresent in the host genome, will include one or more regulatory orfunctional elements which differ from native copies of the gene.

Transformation construct: A chimeric DNA molecule which is designed forintroduction into a host genome by genetic transformation. Preferredtransformation constructs will comprise all of the genetic elementsnecessary to direct the expression of one or more exogenous genes.Transformation constructs prepared in accordance with the instantinvention will include an actin 2 intron and/or an actin 2 promoter. Inparticular embodiments of the instant invention, it may be desirable tointroduce a transformation construct into a host cell in the form of anexpression cassette.

Transformed cell: A cell the DNA complement of which has been altered bythe introduction of an exogenous DNA molecule into that cell.

Transgene: A segment of DNA which has been incorporated into a hostgenome or is capable of autonomous replication in a host cell and iscapable of causing the expression of one or more cellular products.Exemplary transgenes will provide the host cell, or plants regeneratedtherefrom, with a novel phenotype relative to the correspondingnon-transformed cell or plant. Transgenes may be directly introducedinto a plant by genetic transformation, or may be inherited from a plantof any previous generation which was transformed with the DNA segment.

Transgenic plant: A plant or progeny plant of any subsequent generationderived therefrom, wherein the DNA of the plant or progeny thereofcontains an introduced exogenous DNA segment not originally present in anon-transgenic plant of the same strain. The transgenic plant mayadditionally contain sequences which are native to the plant beingtransformed, but wherein the “exogenous” gene has been altered in orderto alter the level or pattern of expression of the gene.

Transit peptide: A polypeptide sequence which is capable of directing apolypeptide to a particular organelle or other location within a cell.

Vector: A DNA molecule capable of replication in a host cell and/or towhich another DNA segment can be operatively linked so as to bring aboutreplication of the attached segment. A plasmid is an exemplary vector.

XII. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Useful expression of transgenes in maize is dependent on the promoterused. The number of effective promoters available for use withtransgenes in maize is not abundant. New promoters, especially promotersthat will express differentially in maize tissues, are spatially and/ortemporally expressed or are induced to express by differentenvironmental signals, would be useful. Such expression specificpromoters could be useful in minimizing yield drag and other potentialadverse physiological effects on maize growth and development that mightbe encountered by constitutive expression of a transgenic protein in aplant. For example, a corn rootworm resistance coding region under thecontrol of a root-specific promoter and disease resistance codingregions under the control of a promoter induced by pathogen attack,would be especially useful. A wider range of effective promoters wouldalso make it possible to introduce multiple transgenes into a plant,each fused to a different promoter, minimizing the risk of DNA sequencehomology dependent transgene activation (co-suppression).

It is known that, often, expression of a transgene, fused to a plantpromoter, does not achieve high levels of expression unless used inconjunction with an intron. Furthermore, not all promoter introncombinations are effective (Simpson and Flipowicz, 1996 ).

The inventors have demonstrated the functionality of a novel isolatedpromoter, preferably in conjunction with an intron in transgenic maize.Designated RS81, this promoter was isolated from a maize B73 genomiclibrary and fused to the gus reporter gene, with and without a modified(internal deletion) rice Actin 2 intron 1. The RS81 promoter is thepromoter of genes expressed in maize root tissue but not in kerneltissue and, in molecular analysis, was shown to have a root-specificexpression profile. Transient expression assays in microparticlebombarded maize suspension cells and in excised maize root and leaftissue, were carried out to determine the functionality of the promoter.The promoter was functionally active, when used in conjunction with amodified (internal deletion) rice Actin 2 intron 1. Furthermore, theRS81 promoter-intron combination achieved transient expression levelsthat are at least 56% the expression level of the strong Actin 1promoter-Actin 1 intron combination (Zhang et al., 1991).

cDNAs that are expressed in maize line B73 root tissue, but not inkernel tissue, were isolated by differential Southern hybridization.Individual bacterial clones from a B73 root cDNA library, were bound toduplicate nitrocellulose membranes. One membrane was hybridized with32P-labeled reverse transcribed B73 poly(A+)RNA from root tissue, andthe second membrane hybridized with ³²P-labeled reverse transcribed B73poly(A+)RNA from kernel tissue. A cDNA that hybridized to root probe,but not to kernel probe were considered for further analysis.

Example 1 Cloning of the RS81 Promoter

The root positive, kernel negative cDNAs were further analyzed bySouthern and Northern analysis. cDNA that remained of interest, waspartially sequenced, and the sequence used the search Genbank forsimilar sequences. Promoter isolation was initiated for a cDNA that wasconsidered proprietary, giving rise to RS81. The RS81 was fullysequenced and used to search Genbank. RS81 cDNA sequence (98.8% completecDNA sequence) had 81% identity to a Orysa saliva root EST (dbj(D23748),68% nucleotide identity to a Hordeum vulgare mRNA for y-TIP-like protein(emb(X80266) and 72% amino acid identity to a Nicotiana tabacum rootspecific tonoplast protein (sp(P30571).

A patent has been issued for the promoter of the RS81-like gene intobacco (U.S. Pat. No. 5,459,252). The RS81 promoter from maize (2584bp) has no significant identity to the tobacco promoter, however.

The root positive, kernel negative cDNAs were used to probe a lambda B73genomic library. Putative clones were isolated through multiple roundsof screening until single clone purity was achieved. DNA from each ofthe lambda clones was characterize by restriction digest analysis andSouthern blot comparison to the banding fingerprint of B73 genomic DNA.A lambda clone matching the genomic pattern was use for isolation of thesequence 5′ of the cDNA start codon. An approximately 4000 bp SalIfragment was cloned into pUC 19. The structure of the RS81 5′ regionobtained is given in FIG. 1 and the corresponding nucleotide sequence isgiven in SEQ ID NO: 1.

Example 2 Cloning of the Rice Actin 2 Intron

Two plasmid clones (pUC-RAc2 and pUC-RAc4) containing genomic DNAsequences of the rice actin 2 gene (Act2) were isolated as described inReece et al., 1990. Restriction maps were generated for each clone,which consisted of an EcoRI restriction fragment from rice genomic DNAcloned in pUC13. The location of the region 5′ of the translation startcodon for in each of these clones was determined by comparing theirrestriction maps with those determined by sequence analysis of thepUC-Rac2 coding region (Reece et al., 1990). Restriction mapping andpreliminary sequence analysis indicated that the pUC-Rac4 genomic clonewas identical to, but 1.2 kb longer than, that of the pUC-Rac2 clone.

Sequence characterization was carried out to determine the length andstructure of the 5′ region in pUC-RAc2 and pUC-RAc4 by the dideoxy chaintermination method using a Perkin-Elmer ABI377 automated sequencingmachine. The sequence analysis of pUC-RAc4 revealed that the genomicclone contained a 2635 bp sequence 5′ of the Act2 translation startcodon (Rac2=1435 bp). In order to determine the structure of the Act2 5′region, a search was carried out of the Rice Genome Project's ESTdatabase with Act2 5′ sequences. This sequence similarity searchidentified a partial cDNA sequence from rice callus tissue (D15626) thatcontains sequences identical to the two transcribed but untranslatedexons, exon 1 and exon 2, in the 5′ region of the Act2 genomic clone. Analignment between the sequence of the Act2 5′ region and the rice ESTwas used to determine the structure of the Act2 5′ sequence. The 2635 bpsequence of the Act2 5′ region was found to be composed of a promoterregion of at least 740 bp, a 5′ transcribed but untranslated first exonof at least 130 bp, a 5′ intron of 1755 bp and the 14 bp transcribed butuntranslated part of the second exon adjacent to the Act2 protein'stranslation initiation site (FIG. 3, SEQ ID NO:3). The 5′ introncontains a ˜300 bp mini transposable element (MITE) of the Tourist (C)type. The Act2 sequence in pUC-RAc2 contains 1.45 kb of Act2 5′ sequenceand starts just upstream of the potential Tourist element within theAct2 5′ intron. The wild type, modified and maize consensus sequencesare as given below, and in SEQ ID NO:6-SEQ ID NO: 11.

Wild-type Act2 5′ intron splice junctions and start codon regionCTGCAGCCGCCATCCCCGGTTCTCTCCTCTTCTTTAG/gtgagcaa PstI Modified Act 5′intron splice junction and start codon region:CTGCAGCTGCCATCCCCGGTTCTCTCCTCTTCTTTAG/gtaaccaa PstIPvuII                              BstEII Zea mays consensus intronsplice junction:                                AG/gtaagtnn Wild-typeAct2 3′ intron splice junctions and start codon region:tttgtgttatgcag/ATCAGTTAAAATAAATGG Modified Act 3′ intron splice junctionand start codon region: ttttttttttgcag/GTCGACTAGGTACCATGG                      SalI   KpnI  NcoI Zea mays consensus 3′ intronsplice junction and start codon region:ttttttttttgcag/GT                ACAATGG

Sequence analysis of the cloned actin 2 intron sequence revealed aTourist-like transposable element within the intron. In order toevaluate the effect of this repetitive element on the function of theactin 2 intron, a modified deletion derivative of the Act2 intron(ΔAct2int), that lacks the inverted repeat sequence associated with theTourist-like transposable element was prepared (SEQ ID NO:5). A 4.3 kBEcoRI-XbaI restriction fragment, containing the Act2 promoter, exon 1,intron 1 and exon 2, was isolated from pUC-RAc4 and cloned intopBSII-SK(−) (Strategene) to create the vector pBS-5′RAc2. A 4.3 kBSalI-SacII restriction fragment, containing the Act2 promoter, exon 1,intron 1 and exon 2, was isolated from pBS-5′RAc2 and cloned intopGEM5Zf(+) (Promega) to create the vector pGEM-5′RAc2. PCR-mediatedsequence mutagenesis was used to introduce KpnI and NcoI restrictionsites around the gus translation initiation codon creating pGEM-PrAct2.pGEM-PrLAct2 was digested with BglII and BclI the intervening intronsequence, containing the Tourist mini-transposon-like inverted repeat,was excised, and the remaining sequence self-ligated to createpGEM-PrAct2Δi.

Example 3 Construction of RS81 Promoter-gus-nos, RS81Promoter-Intron-gus-nos and RS81 Promoter-Intron-gus-nos Fusion Vectors

Restriction enzyme analysis and Southern blot analysis of the lambdaclone indicated that the RS81 promoter region was located on anapproximately 4000 bp SalI gDNA fragment. The SalI fragment was clonedinto pUC19. The bases around the ATG start of the coding region werePCR™ modified to an NcoI restriction site to accommodate fusing thepromoter to the gus gene. The PCR™ fragment contains a HindIII site 5′of the NcoI site. The promoter was then fused to the gus gene in plasmidvector pUC19 in three steps. First, the PCR™ generated NcoI-HindIII 3′end of the promoter was ligated to the NcoI-HindIII site of the gus-nosterminator containing plasmid vector, pGN73. Second, the promoterregion-gus-nos fragment was isolated as a HindIII-EcoRI fragment andligated with the HindIII-PstI fragment (the mid promoter region) intothe PstI-EcoRI site of plasmid vector pUC19. Lastly, the PstI—PstI 5′fragment of the promoter region was ligated into the PstI site of theconstruct and the correct orientation determined. To construct the RS81promoter-intron-gus-nos fusion vector, the NcoI to NruI region (19 bpfrom the ATG start site) at the promoter 3′ end was replaced with theAct2 Δ intron, as a NcoI-PvuII fragment. A RS81 promoter-Act2 Δintron-gus-rbcS fusion vector also was constructed by cloning the RS81promoter-Act2 Δ intron fragment into the gus-rbcS plasmid vector, pGR73.These constructs were then analyzed for expression profiles againstconstructs employing the actin 1 promoter in transient assays asdescribed in Example 4 and Tables 8 and 9.

Example 4 Expression Analysis of Transformation Constructs in TransgenicCells

Transient expression assays were performed as described by Jefferson(1987). Histochemical staining and fluorometric analysis for expressionof the GUS reporter gene (E coli β-glucuronidase), fused to the RS81promoter, was performed using 1×6 (716) callus suspension cells andexcised maize root and leaf tissue. Equal molar concentrations ofplasmid DNA were precipitated onto 0.6 micron gold particles andintroduced into the tissues by microprojectile bombardment. For thehistochemical staining, bombarded tissues were incubated forapproximately 40 h at 24° C., followed by incubation in bufferedsolution containing 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-GLUC),overnight at 37° C. Comparison of expression levels from the RS81promoter-reporter gene constructs, to levels from the Actin 1promoter-Actin 1 intron-reporter gene construct, were visually assessed.For the fluorometric analysis, bombarded suspension callus cells wereincubated for approximately 40 h at 24° C., then scraped off the filterpaper supports and either stored at −70° C. or lysed immediately. 50microliters of cell lysate was incubated in a total volume of 500microliters reaction solution containing4-methyl-umbelliferyl-b-D-glucuronide (MUG). The rate of the enzymaticconversion of MUG (substrate) to 4-methylumbelliferone 4-MU (product),was monitored at 20 min intervals for at least 1.5 h. Fifty microliteraliquots of the reaction mix were removed from the mix at theappropriate interval and the reaction stopped by addition to 0.95milliliters of 0.2M Na₂CO₃. Fluorescence emitted from the samples wasmeasured at emission 365 nm and excitation 455 nm. The readings werenormalized for protein content using the Bradford assay and convertedinto nanomoles of 4-MU produced/μg protein/hour. The results of theassays are given in Tables 8 and 9.

TABLE 8 Promoter activity in transient assays of microparticle bombardedmaize cells: in situ histochemical staining of gus reporter geneexpression Relative GUS Relative Relative Activity in GUS GUS Repor-Suspension Activity in Activity in ter 3′ Culture Leaf Root Pr. IntronGene Term. Cells Tissue Tissue — — gus — − NA NA — — gus nos − NA NA — —gus rbcS − NA NA Act1 Act1 gus — + NA NA Act1 Act1 gus nos +++ +++ +++Act1 Act1 gus rbcS ++ NA NA RS81 — gus nos + − − RS81 Act2Δi gus nos +++++++ +++ RS81 Act2Δi gus rbcS ++++ ++++ ++++ NA = Not Assayed

TABLE 9 Promoter activity in transient assays of microparticle bombardedmaize cells: quantitative fluorometric analysis of GUS specific activityMean Gus Specific Reporter Activity S.E. Pr. Intron Gene 3′ Term. (nMMUG/h/μg protein) (n=2) — — gus — 0.00 9.53 — — gus nos 13.44 1.28 — —gus rbcS 0.34 2.79 Act1 Act1 gus — 115.05 5.79 Act1 Act1 gus nos 843.771.97 Act1 Act1 gus rbcS 304.63 8.34 RS81 — gus nos 10.99 2.34 RS81Act2Δi gus nos 475.95 9.53 RS81 Act2Δi gus rbcS 310.56 9.65

Example 5 Preparation of Microprojectiles

Microprojectiles were prepared by adding 60 mg of 0.6 μm gold particles(BioRad, cat. no. 165-2262) to 1000 μl absolute ethanol and incubatingfor at least 3 hours at room temperature followed by storage at −20° C.Twenty to thirty five μl of the sterile gold particles or morepreferably 30 to 35 μl of gold particles (30 μl contains 1.8 mg ofparticles) were centrifuged in a microcentrifuge for up to 1 min. Thesupernatant was removed and one ml sterile water was added to the tube,followed by centrifugation at 1800-2000 rpm for 2-5 minutes.Microprojectile particles were resuspended in 30 μl of DNA solutioncontaining about 500 ng of vector DNA.

Two hundred twenty microliters sterile water, 250 μl 2.5M CaCl₂ and 50μl stock spermidine (14 μl spermidine in 986 μl water) were then addedto the particle containing solution. The solution was then thoroughlymixed and placed on ice, followed by vortexing at 4° C. for 10 minutesand centrifugation at 500 rpm for 5 minutes The supernatant was removedand the pellet resuspended in 600 μl absolute ethanol. Followingcentrifugation at 500 rpm for 5 minutes, the pellet was resuspended in36-38 μl of absolute ethanol, vortexed for approximately 20 seconds, andsonicated for 20-30 seconds. At this stage the particles were typicallyallowed to sit for 2-5 minutes, after which 5-10 μl of the supernatantwas removed and dispensed on the surface of a flyer disk and the ethanolwas allowed to dry completely. Alternatively, particles may be removeddirectly after resuspension and vortexing 20 to 30 seconds in 36 μl-38μl of ethanol, placed on the flyer disk and allowed to dry as done forthe settled treatment. The bombardment chamber was then evacuated toapproximately 28 in. Hg prior to bombardment. The particles were thenused for bombardment by a helium blast of approximately 1100 psi usingthe DuPont Biolistics PDS1000He particle bombardment device.

Example 6 Bombardment of Hi-II Immature Embryos

Immature embryos (1.2-3.0 mm in length) of the corn genotype Hi-II wereexcised from surface-sterilized, greenhouse-grown ears of Hi-II 10-12days post-pollination. The Hi-II genotype was developed from an A188×B73cross (Armstrong et al., 1991). Approximately 30 embryos per petri dishwere plated axis side down on a modified N6 medium containing 1 mg/l2,4-D, 100 mg/l casein hydrolysate, 2.9 g/L L-proline, 16.9 mg/L silvernitrate, 2 mg/L L-glycine, and 2% sucrose solidified with 2 g/l Gelgro,pH 5.8 (#201V medium). Embryos were cultured in the dark for two to sixdays at 24° C.

Approximately 3-4 hours prior to bombardment, embryos were transferredto the above culture medium with the sucrose concentration increasedfrom 3% to 12%. When embryos were transferred to the high osmoticummedium they were arranged in concentric circles on the plate, starting 1cm from the center of the dish, positioned such that their coleorhizalend was orientated toward the center of the dish. Usually two concentriccircles were formed with 25-35 embryos per plate.

The plates containing embryos were placed on the third shelf from thebottom, 5 cm below the stopping screen. The 1100 psi rupture discs wereused for bombardment. Each plate of embryos was bombarded once with theDuPont Biolistics PDS1000He particle gun. Following bombardment, embryoswere allowed to recover on high osmoticum medium (#201SV, 12% sucrose)overnight (16-24 hours) and were then transferred to selection mediumcontaining 1 mg/l bialaphos (plus I mg/l bialaphos). Embryos weremaintained in the dark at 24° C. After three to four weeks on theinitial selection plates about 90% of the embryos typically formed TypeII callus and were transferred to selective medium containing 3 mg/lbialaphos (#201D). Southern or PCR analysis can then be used foranalysis of transformants and assays of gene expression may be carriedout.

Transformed callus is maintained on medium #204D. Regeneration of plantsis initiated by transfer of callus to MS medium containing 0.04 mg/L NAAand 3 mg/L BAP (medium #105). Tissue is cultured in the dark for twoweeks, followed by two weeks of culture on fresh medium #105 in lowlight. Tissue is subsequently transferred to MS medium with 6% sucrosewithout growth regulators (medium #110) and cultured in low light.Tissue is subcultured once in Petri dishes, followed by two subcultureson #110 medium in PHYTATRAYS. Regenerated plants are transferred fromPHYTATRAYS to soil and grown further in a growth chamber or greenhouse.Tissue in PHYTATRAYS is grown under high light in a growth chamber.

Example 7 Transformation of H99 Immature Embryos or Callus and Selectionwith Paromomycin

Maize immature embryos (1.2-3.0 mm, 10-14 days post pollination) areisolated from greenhouse grown H99 plants that have been self or sibpollinated and are cultured on 735 medium in the dark at approximately27° C. The immature embryos are either bombarded 1-6 days afterisolation or cultured to produce embryogenic callus that is used forbombardment. Embryogenic callus is expanded and maintained bysubculturing at 2-3 week intervals to fresh 735 medium. Prior tobombardment, cultured embryos or embryogenic callus (subdivided inapproximately 2-4 mm clumps) are transferred to 735 medium containing12% sucrose for 3-6 hours. Following bombardment, carried out asdescribed in Example 6, tissue cultures are incubated overnight andtransferred to 735 medium containing 500 mg/L paromomycin. After 2-3weeks, callus is subdivided into small pieces (approximately 2-4 mm indiameter) and transferred to fresh selection medium. This subculturestep is repeated at 2-3 week intervals for up to about 15 weekpost-bombardment, with subdivision and visual selection for healthy,growing callus.

Paromomycin tolerant callus is transferred to 735 medium without 2,4-Dbut containing 3.52 mg/L BAP for 3-9 days in the dark at approximately27° C. and is subsequently transferred to 110 medium (½X MS salts, 0.5mg/L thiamine, 0.5 mg/L nicotinic acid, 3% sucrose, 3.6 g/L Gelgro, pH5.8) containing 100 mg/L paromomycin in Phytatrays (Sigma) and culturedat about 27° C. in the light. Plantlets that develop in Phytatrays after3-6 weeks are then transferred to soil. Plantlets are acclimated in agrowth chamber and grown to maturity in the greenhouse.

Example 8 Methods for Microprojectile Bombardment

Many variations in techniques for microprojectile bombardment are wellknown in the art and therefore deemed useful with the current invention.Exemplary procedures for bombardment are discussed in, for example, U.S.patent appl. Ser. No. 08/113,561, filed Aug. 25, 1993, specificallyincorporated herein by reference in its entirety. Examples of targettissues which may be used with the current invention include immatureembryos, Type I callus, Type II callus, Type III callus, suspensioncultures and meristematic tissue (PCT Publication WO 96/04392). Somegenotypes which are especially useful for maize transformation arespecifically disclosed herein above, as well as in, for example, U.S.patent appl. Ser. No. 08/113,561, filed Aug. 25, 1993. Preferredgenotypes will be those which are readily transformable and which alsomay be regenerated to yield a fertile transgenic plant.

Any method for acceleration of microprojectiles may potentially be usedto transform a plant cell with the current invention. A preferred methodwill be a gas-driven particle gun such as the DuPont BiolisticsPDS1000He particle bombardment device. Exemplary particles forbombardment include those comprised of tungsten, gold, platinum, and thelike. Gold particles are deemed particularly useful in the currentinvention, with 0.6 μm or 0.7 μm gold particles being preferred and 0.6μm particles most preferred. The most preferred particles will be DNAcoated and have a mean size between 0.6 μm and 1.0 μm. Alternatively,particles may be allowed to settle for 2-5 minutes followingprecipitation of DNA onto particles. Particles are then removed from thesupernatant and used for microprojectile bombardment. It is believedthat the settling step enriches for a population of particles coatedwith DNA in which fewer aggregates of particles are present.

As disclosed herein, any DNA sequence may potentially be used fortransformation. The DNA segments used for transformation will preferablyinclude one or more selectable, secretable or screenable markers. Manyexamples of such are well known in the art and are specificallydisclosed herein. In the case of selectable markers, selection may be insolid or liquid media. The DNA segments used will preferably alsoinclude one or more genes which confer, either individually or incombination with other sequences, a desired phenotype on the transformedplant. Exemplary genes for transformation and the correspondingphenotypes these sequences may confer on the transformed plant aredisclosed herein.

Example 9 Expression Profile Analysis of the RS81 Promoter in StablyTransformed Plants

Expression cassettes comprising the RS81 promoter-Act2Δintron-GUS-nos(plasmid vector pDPG894) and RS81 promoter-Act2Δintron-GUS-rbcS(pDPG895) were introduced separately into HiII immature embryos asdescribed in Examples 5 and 6. Each plasmid was bombarded into HiIIimmature embryos in conjunction with pDPG16, comprising a 35Spromoter-bar-Tr7 expression cassette. Transformants were selected asdescribed in Example 6. Expression of the GUS gene was assayed asdescribed in Example 4 and Jefferson (1987).

Three of twenty-one transformants that were tested, comprising the RS81promoter-Act2Δintron-GUS-rbcS expression cassette, expressed the GUSgene in callus. Plants were regenerated from each of the RS81 promoter-Act2Δintron-GUS-rbcS transformants. Root and leaf material werecollected from plantlets grown in vitro at two times duringregeneration. Plantlets were first assayed during the second month ofplant regeneration. Plantlets from nine of the fifteen transformantsexpressed the GUS gene in roots, and five transformants that expressedGUS in roots also expressed GUS in the regenerating stem and leafmaterial. Plantlets also were assayed during the third month of plantregeneration, close to the time of transfer of plantlets to soil. GUSexpressed in roots of twelve of eighteen transformants that wereassayed. Furthermore, eight of the twelve plantlets that expressed GUSin roots also expressed GUS in stem and leaf material. An additional twoplantlets expressed GUS in stem and leaf material, but did not expressGUS in roots. GUS expression was observed in roots or R₀ plants growingin soil in the greenhouse that were regenerated from fourteentransformants. GUS expression was further assayed in seed harvested fromR₀ plants derived from four transformants. In three transformants, GUSexpression was observed in the seed.

One transformant, of eighteen assayed, that comprised the RS8 1promoter- Act2Δintron-GUS-nos expression cassette, expressed the GUSgene in callus. Plants were regenerated from thirteen transformants.Root and leaf material were collected from regenerating plantlets grownin vitro and two times during regeneration. Plantlets, assayed duringthe second month of plant regeneration, from ten of the fourteentransformants, expressed GUS in roots. Only two transformants comprisingthe RS8 1 promoter and the nos terminator expressed GUS in roots and inregenerating shoot and leaf material. This observation suggests that theterminator sequence, e.g., rbcS, may influence the tissue specificity ofgene expression. Plantlets assayed during the third month ofregeneration, close to the time of transfer of plantlets to soil,expressed GUS in roots of five or six transformants assayed. Three ofthe five root expressing transformants also expressed GUS in stem andleaf material. Plants from fifteen transformants were transferred tosoil in the greenhouse.

Example 10 Histological Analysis of the RS81 Promoter in StablyTransformed Plants

Immature embryos of the Hill genotype were bombarded and plantsregenerated as described in Example 6 with a vector comprising the bargene (pDPG165) and a second vector comprising the RS81 promoter operablylinked to exon 1 of the rice actin 2 gene, the intron 1 of the riceactin 2 gene, the uidA gene encoding beta-glucuronidase and the 3′region derived from the Agrobacterium tumefaciens nopaline synthase gene(pDPG894). Root tissue was harvested from a greenhouse grown R₀ plantregenerated from a transformant designated II072SZ51202. Expression ofthe GUS gene was assayed as described in Example 4 and Jefferson (1987).Tissue samples were incubated in the GUS assay mix overnight at 37° C.Tissue was then transferred to FAA fixative (4% formalin, 5% aceticacid, 50% ethanol) at 0° C. The FAA was removed and replaced with freshcold FAA, followed by incubation of the tissues at 4° C. overnight. FAAwas replaced by 50% ethanol followed by dehydration by sequentialtransfer to 70%, 85%, 90% and twice in 100% ethanol. Tissue samples wereinfiltrated with Histoclear (National Diagnostics) by incubation in 1:1ethanol:Histoclear followed by 3:1 Histoclear:ethanol, and twice inHistoclear. Molten Paraplast X-Tra (Oxford Labware) embedding wax wasadded. After two wax changes, tissue was embedded, trimmed and mountedfor sectioning. Forty micron sections were cut on a Reichert-Jung rotarymicrotome and fixed to ProbeOn plus slides (Fisher Scientific). Sectionswere deparafinized in Histoclear and mounted directly or counterstainedwith Safranin O (in 70% ethanol) for 15 minutes, followed by Histoclear.Slides were sealed with HistoResin (Fisher Scientific) and visualizedusing 50× bright field optics on a Zeiss Axiophot (Germany) modelEL-Einsatz 451888 microscope. Images were captured on Kodak Ektachrome160 Tungsten film. The results of this study, as seen in FIG. 4,revealed expression of GUS directed by the RS81 promoter in the rootendodermis surrounding the vascular bundles. This pattern of expressionis consistent with RS81 being a promoter of a gene involved in watermovement. Also, the coding sequence of the RS81 gene has homology toHordeum vulgare and N. tabacum genes for tonoplast intrinsic proteins,which are aquaporin-like proteins involved in water movement within theplant.

Example 11 Introgression of Transgenes Into Elite Inbreds and Hybrids

Backcrossing can be used to improve a starting plant. Backcrossingtransfers a specific desirable trait from one source to an inbred orother plant that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriategene(s) for the trait in question, for example, a construct prepared inaccordance with the current invention. The progeny of this cross firstare selected in the resultant progeny for the desired trait to betransferred from the non-recurrent parent, then the selected progeny aremated back to the superior recurrent parent (A). After five or morebackcross generations with selection for the desired trait, the progenyare hemizygous for loci controlling the characteristic beingtransferred, but are like the superior parent for most or almost allother genes. The last backcross generation would be selfed to giveprogeny which are pure breeding for the gene(s) being transferred, i.e.one or more transformation events.

Therefore, through a series a breeding manipulations, a selectedtransgene may be moved from one line into an entirely different linewithout the need for further recombinant manipulation. Transgenes arevaluable in that they typically behave genetically as any other gene andcan be manipulated by breeding techniques in a manner identical to anyother corn gene. Therefore, one may produce inbred plants which are truebreeding for one or more transgenes. By crossing different inbredplants, one may produce a large number of different hybrids withdifferent combinations of transgenes. In this way, plants may beproduced which have the desirable agronomic properties frequentlyassociated with hybrids (“hybrid vigor”), as well as the desirablecharacteristics imparted by one or more transgene(s).

Example 12 Marker Assisted Selection

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

In the process of marker assisted breeding, DNA sequences are used tofollow desirable agronomic traits (Tanksley et al., 1989) in the processof plant breeding. Marker assisted breeding may be undertaken asfollows. Seed of plants with the desired trait are planted in soil inthe greenhouse or in the field. Leaf tissue is harvested from the plantfor preparation of DNA at any point in growth at which approximately onegram of leaf tissue can be removed from the plant without compromisingthe viability of the plant. Genomic DNA is isolated using a proceduremodified from Shure et al. (1983). Approximately one gram of leaf tissuefrom a seedling is lypholyzed overnight in 15 ml polypropylene tubes.Freeze-dried tissue is ground to a powder in the tube using a glass rod.Powdered tissue is mixed thoroughly with 3 ml extraction buffer (7.0urea, 0.35M NaCl, 0.05M Tris-HCl pH 8.0, 0.01M EDTA, 1% sarcosine).Tissue/buffer homogenate is extracted with 3 ml phenol/chloroform. Theaqueous phase is separated by centrifugation, and precipitated twiceusing {fraction (1/10)} volume of 4.4M ammonium acetate pH 5.2, and anequal volume of isopropanol. The precipitate is washed with 75% ethanoland resuspended in 100-500 μl TE (0.01M Tris-HCl, 0.001M EDTA, pH 8.0).

Genomic DNA is then digested with a 3-fold excess of restrictionenzymes, electrophoresed through 0.8% agarose (FMC), and transferred(Southern, 1975) to Nytran (Schleicher and Schuell) using 10×SCP (20SCP: 2M NaCl, 0.6M disodium phosphate, 0.02M disodium EDTA). The filtersare prehybridized in 6×SCP, 10% dextran sulfate, 2% sarcosine, and 500μg/ml denatured salmon sperm DNA and ³²P-labeled probe generated byrandom priming (Feinberg & Vogelstein, 1983). Hybridized filters arewashed in 2×SCP, 1% SDS at 65° C. for 30 minutes and visualized byautoradiography using Kodak XAR5 film. Genetic polymorphisms which aregenetically linked to traits of interest are thereby used to predict thepresence or absence of the traits of interest.

Those of skill in the art will recognize that there are many differentways to isolate DNA from plant tissues and that there are many differentprotocols for Southern hybridization that will produce identicalresults. Those of skill in the art will recognize that a Southern blotcan be stripped of radioactive probe following autoradiography andre-probed with a different probe. In this manner one may identify eachof the various transgenes that are present in the plant. Further, one ofskill in the art will recognize that any type of genetic marker which ispolymorphic at the region(s) of interest may be used for the purpose ofidentifying the relative presence or absence of a trait, and that suchinformation may be used for marker assisted breeding.

Each lane of a Southern blot represents DNA isolated from one plant.Through the use of multiplicity of gene integration events as probes onthe same genomic DNA blot, the integration event composition of eachplant may be determined. Correlations may be established between thecontributions of particular integration events to the phenotype of theplant. Only those plants that contain a desired combination ofintegration events may be advanced to maturity and used for pollination.DNA probes corresponding to particular transgene integration events areuseful markers during the course of plant breeding to identify andcombine particular integration events without having to grow the plantsand assay the plants for agronomic performance.

It is expected that one or more restriction enzymes will be used todigest genomic DNA, either singly or in combinations. One of skill inthe art will recognize that many different restriction enzymes will beuseful and the choice of restriction enzyme will depend on the DNAsequence of the transgene integration event that is used as a probe andthe DNA sequences in the genome surrounding the transgene. For a probe,one will want to use DNA or RNA sequences which will hybridize to theDNA used for transformation. One will select a restriction enzyme thatproduces a DNA fragment following hybridization that is identifiable asthe transgene integration event. Thus, particularly useful restrictionenzymes will be those which reveal polymorphisms that are geneticallylinked to specific transgenes or traits of interest.

Example 13 General Methods for Assays

DNA analysis of transformed plants is performed as follows. Genomic DNAis isolated using a procedure modified from Shure et al., 1983.Approximately 1 gm callus or leaf tissue is ground to a fine powder inliquid nitrogen using a mortar and pestle. Powdered tissue is mixedthoroughly with 4 ml extraction buffer (7.0M urea, 0.35M NaCl, 0.05MTris-HCl pH 8.0, 0.01M EDTA, 1% sarcosine). Tissue/buffer homogenate isextracted with 4 ml phenol/ chloroform. The aqueous phase is separatedby centrifugation, passed through Miracloth, and precipitated twiceusing {fraction (1/10)} volume of 4.4M ammonium acetate, pH 5.2 and anequal volume of isopropanol. The precipitate is washed with 70% ethanoland resuspended in 200-500 μl TE (0.01M Tris-HCl, 0.001M EDTA, pH 8.0).

The presence of a DNA sequence in a transformed cell may be detectedthrough the use of polymerase chain reaction (PCR). Using this techniquespecific fragments of DNA can be amplified and detected followingagarose gel electrophoresis. For example, two hundred to 1000 ng genomicDNA is added to a reaction mix containing 10 mM Tris-HCl pH 8.3, 1.5 mMMgCl₂, 50 mM KCl, 0.1 mg/ml gelatin, 200 μM each dATP, dCTP, dGTP, dTTP,0.5 μM each forward and reverse DNA primers, 20% glycerol, and 2.5 unitsTaq DNA polymerase. The reaction is run in a thermal cycling machine asfollows: 3 minutes at 94° C., 39 repeats of the cycle 1 minute at 94°C., 1 minute at 50° C., 30 seconds at 72° C., followed by 5 minutes at72° C. Twenty μl of each reaction mix is run on a 3.5% NuSieve gel inTBE buffer (90 mM Tris-borate, 2 mM EDTA) at 50V for two to four hours.Using this procedure, for example, one may detect the presence of thebar gene, using the forward primer CATCGAGACAAGCACGGTCAACTTC (SEQ IDNO:12) and the reverse primer AAGTCCCTGGAGGCACAGGGCTTCAAGA (SEQ ID NO:13). Primers for the Act2 intron or RS81 promoter can be readilyprepared by one of skill in the art in light of the sequences given inSEQ ID NO:3 and SEQ ID NO: 1, respectively.

A method to detect the presence of phosphinothricin acetyl transferase(PAT) involves the use of an in vitro enzyme reaction followed by thinlayer chromatography, as described in U.S. patent appl. Ser. No.08/113,561, filed Aug. 25, 1993 (specifically incorporated herein byreference in its entirety). The procedure is conducted by preparingvarious protein extracts from homogenates of potentially transformedcells, and from control cells that have neither been transformed norexposed to bialaphos selection, and then assaying by incubation with PPTand ¹⁴C-Acetyl Coenzyme A followed by thin layer chromatography. Theresults of this assay provide confirmation of the expression of the bargene which codes for phosphinothricin acetyl transferase (PAT).

For Southern blot analysis genomic DNA is digested with a 3-fold excessof restriction enzymes, electrophoresed through 0.8% agarose (FMC), andtransferred (Southern, 1975) to Nytran (Schleicher and Schuell) using10×SCP (20×SCP: 2M NaCl, 0.6M disodium phosphate, 0.02M disodium EDTA).Probes are labeled with ³²P using the random priming method (BoehringerMannheim) and purified using Quik-Sep® spin columns (Isolab Inc., Akron,Ohio). Filters are prehybridized at 65° C. in 6×SCP, 10% dextransulfate, 2% sarcosine, and 500 μg/ml heparin (Chomet et al., 1987) for15 min. Filters then are hybridized overnight at 65° C. in 6×SCPcontaining 100 μg/ml denatured salmon sperm DNA and ³²P-labeled probe.Filters are washed in 2×SCP, 1% SDS at 65° C. for 30 min. and visualizedby autoradiography using Kodak XAR5 film. For rehybridization, thefilters are boiled for 10 min. in distilled H₂O to remove the firstprobe and then prehybridized as described above.

Example 14 Utilization of Transgenic Crops

The ultimate goal in plant transformation is to produce plants which areuseful to man. In this respect, transgenic plants created in accordancewith the current invention may be used for virtually any purpose deemedof value to the grower or to the consumer. For example, one may wish toharvest seed from transgenic plants. This seed may in turn be used for awide variety of purposes. The seed may be sold to farmers for plantingin the field or may be directly used as food, either for animals orhumans. Alternatively, products may be made from the seed itself.Examples of products which may be made from the seed include, oil,starch, animal or human food, pharmaceuticals, and various industrialproducts. The food uses of maize, in addition to human consumption ofmaize kernels, include both products of dry- and wet-milling industries.The principal products of maize dry milling are grits, meal and flour.The maize wet-milling industry can provide maize starch, maize syrups,and dextrose for food use. Maize oil is recovered from maize germ, whichis a by-product of both dry- and wet-milling industries.

Maize, including both grain and non-grain portions of the plant, also isused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry. Industrial uses of maize include productionof ethanol, maize starch in the wet-milling industry and maize flour inthe dry-milling industry. The industrial applications of maize starchand flour are based on functional properties, such as viscosity, filmformation, adhesive properties, and ability to suspend particles. Themaize starch and flour have application in the paper and textileindustries. Other industrial uses include applications in adhesives,building materials, foundry binders, laundry starches, explosives,oil-well muds, and other mining applications. Plant parts other than thegrain of maize also are used in industry, for example, stalks and husksare made into paper and wallboard and cobs are used for fuel and to makecharcoal. Other means for utilizing plants, such as those that may bemade with the current invention, have been well known since the dawn ofagriculture and will be known to those of skill in the art in light ofthe instant disclosure. Specific methods for crop utilization may befound in, for example, Sprague and Dudley (1988), and Watson and Ramstad(1987).

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All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe methods described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

13 1 2584 DNA Zea mays modified_base (321)..(2162) N = A or C or G or T1 gtcaacggat cacatagcta gcgtctgatt ccatgtgaaa tacttttttt cccgaacaca 60tgatatcccc attttcattc atttcggatc atttgaattg aattacattc taataattat 120aatttagaca catattaatt aagctaatat gattctatgc ggaatatatt tgtaaatgct 180cgtaatatat tttaagcact gttttagatt taaaagttct ctttcgacct tcagcgttct 240gtcattttgt taaggctaag gtggatctaa agttttgcag ggagctagtg tcaaggaaga 300cctaatactt ccactcacga ngagcaacta ggcaagccac acccttataa aaaaaaggca 360aaccagacag tcananagac aggggctctt ccgccaactg gaccttgatg ttgccagtca 420tgcgctgaaa gattccaaat cctcaatcgc anactgcctt gtcaatcatt ttttgcatta 480aggntaaagc gataaggtaa acaaccattg gartnktkgt taacancanc ctggcacggc 540cggatgcntn ntntgaataa tantnngggg ncaagacagg aanncntaat ntttggaaat 600tttagtggnt aggagtcact gatcatggac ntaagagtgc taacccactg cattancaaa 660aancgtgtgg gtgttgcggn ttccaanttg tccgttgtca cggnatggta tgaatatgcc 720aaagattctc gctgttttgt gaaatttaga tgagctgagc atgacaaaag cgcaaacctt 780aattgcattg cctactccat atatggcaag atggcttgca tgacaaaatt agatgttaac 840aaaaagaaaa caaggagaag gtatagaggt gctcgaaaga ctcgttgaca ctttaaactc 900gggtgtgcgt gtgttatgtg tttatctgct gtattcagaa cggctcgtca taatagcgta 960catatatata tatatatgat ccaattttga aagtactaga taaatataag atactatgca 1020tttaaactat attatagtct tatactctta tcttagaacc ggaaccgtcc tatacattat 1080ctcttaatag cctctccctc aatcttaatc ttctctaggt ttagattatg gttagatcga 1140attctctact aatcttacga ataaagcttt agtaaatcag ccaaccacnt cattctttng 1200aataaacaaa atcaatctca agcattctta taaaaactct ttctcgaaca aattgataac 1260caaattctat atgctttatt atnggcdtgg aacaaacgaa ttagatnaca aatatntttt 1320gcatatcatg cgcgcgchag tgatgtttta cttttactcn tcggtcacag ggttthgcct 1380ccttgaawtc gaaaggggaa tactgamtgg ccaaattgct tatggagttg ttaaagaata 1440caaaaatagg tgccgaaagg gtttgctcta atgatttgat gaagtcgttg ccmccgatgg 1500ttaccatgca gtactactac tatcatagtg tcatggtttt tttattttca attgctgtac 1560tgatgcagac gtgacgcatg cgtttactat ccttgcagcc accgactcca aaatgaagag 1620agctagcgct caattcattg gtccacgtca ccatcaacgt tccgaactgt ctcgcatgca 1680tcgtgccgag aaaaagagca ccgatgacna tggatgcgat cgadttccca ccatttgaga 1740gcaatatgcg ttttacactt tacaacaagg tacaagtgaa acaataaacc atgctaaata 1800aaagcgctaa gggctgattt ggtggcccag aatcacggga ggattggagg ggattgagag 1860agaaattagt tcatttcctc ctcaatcatc tacaattctc tcgtgattct acttatcacc 1920aaatcaatct aaaacttacg tctctcctgc tgcgctctcg caccacactg caggaacgta 1980cgcccggccg aggctgcaac tgaaatggaa gtatgaaacc cttcggcttt tgtttcctga 2040ttgaccaact gtgacaaatc ccctagcatt ttgcaagaac aactacatag acgttgagat 2100aacatatata tcctccaaac gagacagccc atctgacaac acttgcagcc gttcgaacgt 2160tnccattacc aaaatattta agcgttacac aagagatagc gtacctccaa tgattagcgg 2220ctttgccgtt tattattgtc attggcgcat tgctttggtg tacctcacat ggacacgcca 2280tgaaaagatg acaatagcgc aaacagacat ccgaacaaag cttggtgcgc ctgctctcgg 2340ccgggctagc tagtaccaat catggaataa taagctagta tatatatacc gccccattta 2400taaaaatgta aataaaatgt tctgtggcga ttatgcacac cgtccataat actctagtcg 2460tcggcaagct gaggccgccc attaagcctt gagcgtgtat aaaaggggca tagactccca 2520gcatcagcga agcagcaaca tatcaagctc agagcctcag agcgccagcc aagtcttgcg 2580gtcg 2584 2 329 DNA RICE 2 gggctgcgag gagtctggtg gcacctaagc cgtcatcgtcatatatgcct cgtttaattg 60 ttcatctctg attcgatgat gtctcccacc ttgtttcgtgtgttcccagt ttgttcatcg 120 tcttttgatt ttaccggccg tgctctgctt ttgtttttgtttcacctgat ctctctctga 180 cttgatgtaa gagtggtatc tgctacgact atatgttgttgggtgaggca tatgtgaatg 240 aaatmtatgr aagctccggc tatatatatt tatacaaagggtatgagatg gatgtgaatc 300 tagagcatat gtgtccaaca atcaattcg 329 3 2640 DNARICE modified_base (1094)..(2167) N = A or C or G or T 3 gaattcccggacctccatgc ctacatcaac taatttgatt ccttgagttt acgtttagtg 60 atatgtctatttttagagct tgttggggct tcggcctcag ctctagccag ccaaacatgt 120 tctaccaagtaccctatgtt ggcatgatat agtgatgcat tataacaata aatgagcgag 180 ggattgctggctgaaaaagc tatactagct gcatttggtt atagttaacc gaactattaa 240 ttgcgtgtacaacaaaataa aaaaaatgca tgttgcacat tctttcatta acattatgtt 300 ttggtagtgtgaattagaaa tttgattgac agtagatcga caaacatagt ttcaatatgc 360 ttaagttagttatgacttta acatatcagt ctccttgata ttttcgtttt agattcgtct 420 ctctactagtgtgtatgtcc accttccata gcagtgaagg gttccattcc atccctggta 480 aaaaaaaatcaaccactact atttatttcc taaaaagcaa aatgataaaa tatcattttt 540 ttaataaaaataaaaaaatt ttggggtaca taattgatgt tgccccttgg gattaacctt 600 aaaaaagggcgaattttcta gggtttggcc aagttttgca atgcaccaaa ttattcccct 660 tgggccggccgccaccccaa aaaaaacccc aacccccaac tttccattga aggccgggcc 720 cccttaaatcctcatccccc caatttccac caccatcgcc attgccacca cctctcctat 780 atctcgccctccccctcctc cctcccacgc cattcgcctc cttcttgctg cagccgccat 840 ccccggttcggttctctcct cttctttagg tgagcaactg cctctccatg tccaggccct 900 cccggccccygsktgswtty tgktttaawg skkgakgttt ytkgcaaats ggarrkgttt 960 tmkwtttctgttarrwgggk ggaaawackg aackgarttg ctgaaaktag gkgttggctg 1020 ggtkgcttttggctkgtawg ttgtcaaakg ttggawccgt tggamtgtag gragttcagg 1080 graksscstaaacnggtgtt gtttctgggg gatgctgatc cgatccgatg gcttttagtn 1140 gatggaagtatccgatcttg tttgtgctga ggtgacgagt attcttgcag tagatctttt 1200 tcgtgtttatgttgtgttgt gctaaggtct tgtagttccc aaaatttttt ccccaaaaat 1260 gtcaacatggtatctttaga cacatgaata gagcattaaa tatagattaa aaaaaactaa 1320 ttgcacaatttgcatggaaa atcgtgagac caatctttta agcctaatta gtccatgatt 1380 agacataagtgctacagtaa cccacgtgtg ctaatgatgg attaattagg cttaataaat 1440 tcgtctctcagttttctagg cgagctatga aattaatttt ttttattcgt gtccgaaaat 1500 cccttccgacatccggttaa acgtcggatg tgacaagaaa aattttcttt tcgcgaacta 1560 aacaaggcctaaggcgtgaa gttgggggta tgtttacttt gaattgtaga tcaactgaca 1620 gacttttgcatgctcatagc cggtttgttt gcggtactca agaaactgtc ttgattggtc 1680 attccgtagggtggggactk gkgaaaaagc tgattccttt cttttcattt ccacggttgc 1740 tttcttggttggcgtgggaa aaaaacagtt ttcagtactg taccgatcga ctttcttttg 1800 agacttttttctccttcaac aaaacatttc atagttcaca caaaaacaca agcataccaa 1860 cgatttcattatgtgacatg gcttctaaaa tctgaattaa agaagcaagt tgcttaactg 1920 aaaactgcctagtttcagaa atcatggagt ttaaattttc caaagagaag ggtaacatat 1980 tatggagaactagaattttg ttactaaaaa atgtatgctt atgggaccac tattctaaga 2040 tgcttcacatcttgatgacg gctgtctgat cagaaaaaaa ataatgcttc agatcaacca 2100 atcagacaatccaggatatg agcagatcat gttgcattca ttycatccac tgaagcangt 2160 cccnannttcttcccctgaa gattggtcta aatcgattca taaaacacat tgcatgtatg 2220 cttcttaggagagagcacca ttccctttgg agggttggtg attcagacca gcctcggttg 2280 attgatttgaatttcttaac tacaagtcac ttgatctagt tataatttac gcatcatgga 2340 ccattcattttgggagtttc ctatatacaa ctaaagtgtt atacttcttc ctatctgcgc 2400 cttcctttttgtttgaataa tcctccctct ttcacaattt gcaatactag ttagtcaatt 2460 aatagctttgaatgtgatat cttaaagaca tgtattttgt cattcatgtt tgatgaagac 2520 tcgtgtttttgtaggatgaa tgtttagttc aagttacatt tttctgtatt aatctatagt 2580 ctttgtaaacactgttttga atgatttatt ttgtgttatg cagatcagtt aggtaccatg 2640 4 1763 DNARICE modified_base (234)..(1307) N = A or C or G or T 4 cttctttaggtgagcaactg cctctccatg tccaggccct cccggccccy gsktgswtty 60 tgktttaawgskkgakgttt ytkgcaaats ggarrkgttt tmkwtttctg ttarrwgggk 120 ggaaawackgaackgarttg ctgaaaktag gkgttggctg ggtkgctttt ggctkgtawg 180 ttgtcaaakgttggawccgt tggamtgtag gragttcagg graksscsta aacnggtgtt 240 gtttctgggggatgctgatc cgatccgatg gcttttagtn gatggaagta tccgatcttg 300 tttgtgctgaggtgacgagt attcttgcag tagatctttt tcgtgtttat gttgtgttgt 360 gctaaggtcttgtagttccc aaaatttttt ccccaaaaat gtcaacatgg tatctttaga 420 cacatgaatagagcattaaa tatagattaa aaaaaactaa ttgcacaatt tgcatggaaa 480 atcgtgagaccaatctttta agcctaatta gtccatgatt agacataagt gctacagtaa 540 cccacgtgtgctaatgatgg attaattagg cttaataaat tcgtctctca gttttctagg 600 cgagctatgaaattaatttt ttttattcgt gtccgaaaat cccttccgac atccggttaa 660 acgtcggatgtgacaagaaa aattttcttt tcgcgaacta aacaaggcct aaggcgtgaa 720 gttgggggtatgtttacttt gaattgtaga tcaactgaca gacttttgca tgctcatagc 780 cggtttgtttgcggtactca agaaactgtc ttgattggtc attccgtagg gtggggactk 840 gkgaaaaagctgattccttt cttttcattt ccacggttgc tttcttggtt ggcgtgggaa 900 aaaaacagttttcagtactg taccgatcga ctttcttttg agactttttt ctccttcaac 960 aaaacatttcatagttcaca caaaaacaca agcataccaa cgatttcatt atgtgacatg 1020 gcttctaaaatctgaattaa agaagcaagt tgcttaactg aaaactgcct agtttcagaa 1080 atcatggagtttaaattttc caaagagaag ggtaacatat tatggagaac tagaattttg 1140 ttactaaaaaatgtatgctt atgggaccac tattctaaga tgcttcacat cttgatgacg 1200 gctgtctgatcagaaaaaaa ataatgcttc agatcaacca atcagacaat ccaggatatg 1260 agcagatcatgttgcattca ttycatccac tgaagcangt cccnannttc ttcccctgaa 1320 gattggtctaaatcgattca taaaacacat tgcatgtatg cttcttagga gagagcacca 1380 ttccctttggagggttggtg attcagacca gcctcggttg attgatttga atttcttaac 1440 tacaagtcacttgatctagt tataatttac gcatcatgga ccattcattt tgggagtttc 1500 ctatatacaactaaagtgtt atacttcttc ctatctgcgc cttccttttt gtttgaataa 1560 tcctccctctttcacaattt gcaatactag ttagtcaatt aatagctttg aatgtgatat 1620 cttaaagacatgtattttgt cattcatgtt tgatgaagac tcgtgttttt gtaggatgaa 1680 tgtttagttcaagttacatt tttctgtatt aatctatagt ctttgtaaac actgttttga 1740 atgatttattttgtgttatg cag 1763 5 889 DNA RICE modified_base (225)..(423) N = A or Cor G or T 5 gtaaccaact gcctctccat gtccaggccc tcccggcccc ygsktgswttytgktttaaw 60 gskkgakgtt tytkgcaaat sggarrkgtt ttmkwtttct gttarrwgggkggaaawack 120 gaackgartt gctgaaakta ggkgttggct gggtkgcttt tggctkgtawgttgtcaaak 180 gttggawccg ttggamtgta ggragttcag ggraksscst aaacnggtgttgtttctggg 240 ggatgctgat ccgatccgat ggcttttagt ngatggaagt atccgatcttgtttgtgctg 300 aggtgacgag tattcttgca gtagatcaga aaaaaaataa tgcttcagatcaaccaatca 360 gacaatccag gatatgagca gatcatgttg cattcattyc atccactgaagcangtcccn 420 annttcttcc cctgaagatt ggtctaaatc gattcataaa acacattgcatgtatgcttc 480 ttaggagaga gcaccattcc ctttggaggg ttggtgattc agaccagcctcggttgattg 540 atttgaattt cttaactaca agtcacttga tctagttata atttacgcatcatggaccat 600 tcattttggg agtttcctat atacaactaa agtgttatac ttcttcctatctgcgccttc 660 ctttttgttt gaataatcct ccctctttca caatttgcaa tactagttagtcaattaata 720 gctttgaatg tgatatctta aagacatgta ttttgtcatt catgtttgatgaagactcgt 780 gtttttgtag gatgaatgtt tagttcaagt tacatttttc tgtattaatctatagtcttt 840 gtaaacactg ttttgaatga tttatttttt tttttgcagg tcgactagg 8896 45 DNA Zea mays 6 ctgcagccgc catccccggt tctctcctct tctttaggtg agcaa 457 45 DNA Zea mays 7 ctgcagctgc catccccggt tctctcctct tctttaggta accaa 458 10 DNA Zea mays modified_base (9)..(10) N = A or C or G or T 8aggtaagtnn 10 9 32 DNA Zea mays 9 tttgtgttat gcagatcagt taaaataaat gg 3210 32 DNA Zea mays 10 tttttttttt gcaggtcgac taggtaccat gg 32 11 23 DNAZea mays 11 tttttttttt gcaggtacaa tgg 23 12 25 DNA Streptomyceshygroscopicus 12 catcgagaca agcacggtca acttc 25 13 28 DNA Streptomyceshygroscopicus 13 aagtccctgg aggcacaggg cttcaaga 28

What is claimed is:
 1. An isolated nucleic acid comprising a maize RS81 promoter.
 2. An isolated maize RS81 promoter isolatable from the nucleic acid sequence of SEQ ID NO:1.
 3. The isolated nucleic acid of claim 1, wherein said maize RS81 promoter comprises from about 50 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 4. The isolated nucleic acid of claim 1, wherein said maize RS81 promoter comprises from about 150 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 5. The isolated nucleic acid of claim 1, wherein said maize RS81 promoter comprises from about 250 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 6. The isolated nucleic acid of claim 1, wherein said maize RS81 promoter comprises from about 400 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 7. The isolated nucleic acid of claim 1, wherein said maize RS81 promoter comprises from about 750 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 8. The isolated nucleic acid of claim 1, wherein said maize RS81 promoter comprises from about 1000 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 9. The isolated nucleic acid of claim 1, wherein said maize RS81 promoter comprises the nucleic acid sequence of SEQ ID NO:1.
 10. An expression cassette comprising a maize RS81 promoter operably linked to a selected coding region.
 11. The expression cassette of claim 10, wherein said selected coding region encodes an insect resistance protein, a bacterial disease resistance protein, a fungal disease resistance protein, a viral disease resistance protein, a nematode disease resistance protein, a herbicide resistance protein, a protein affecting grain composition or quality, a nutrient utilization protein, a mycotoxin reduction protein, a male sterility protein, a selectable marker protein, a screenable marker protein, a negative selectable marker protein, an environment or stress resistance gene, or a protein affecting plant agronomic characteristics.
 12. The expression cassette of claim 11, wherein said selected protein is a selectable marker protein selected from the group consisting of phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase and glyphosate oxidoreductase.
 13. An expression vector comprising the expression cassette of claim
 10. 14. The expression vector of claim 13, further defined as a plasmid vector.
 15. The expression vector of claim 14, wherein said plasmid vector is located within a bacterial cell.
 16. A transgenic plant stably transformed with a selected DNA comprising a maize RS81 promoter.
 17. The transgenic plant of claim 16, wherein said maize RS81 promoter is isolatable from the nucleic acid sequence of SEQ ID NO:1.
 18. The transgenic plant of claim 16, wherein said maize RS81 promoter comprises from about 50 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 19. The transgenic plant of claim 16, wherein said maize RS81 promoter comprises from about 100 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 20. The transgenic plant of claim 16, wherein said maize RS81 promoter comprises from about 200 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 21. The transgenic plant of claim 16, wherein said maize RS81 promoter comprises from about 400 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 22. The transgenic plant of claim 16, wherein said maize RS81 promoter comprises from about 750 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 23. The transgenic plant of claim 16, wherein said maize RS81 promoter comprises from about 1000 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 24. The transgenic plant of claim 16, wherein said maize RS81 promoter comprises from about 1500 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 25. The transgenic plant of claim 16, wherein said maize RS81 promoter comprises from about 2000 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 26. The transgenic plant of claim 16, wherein said maize RS81 promoter comprises the nucleic acid sequence of SEQ ID NO:1.
 27. The transgenic plant of claim 16, wherein said s elected DNA further comprises a selected coding region operably linked to said maize RS81 promoter.
 28. The transgenic plant of claim 27, wherein said s elected coding region encodes an insect resistance protein, a bacterial disease resistance protein, a fungal disease resistance protein, a viral disease resistance protein, a nematode disease resistance protein, a herbicide resistance protein, a protein affecting grain composition or quality, a nutrient utilization protein, an environment or stress resistance protein, a mycotoxin reduction protein, a male sterility protein, a selectable marker protein, a screenable marker protein, a negative selectable marker protein, or a protein affecting plant agronomic characteristics.
 29. The transgenic plant of claim 28, wherein said selected protein is a selectable marker protein selected from the group consisting of phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase and glyophosate oxidoreductase.
 30. The transgenic plant of claim 16, wherein said selected DNA comprises plasmid DNA.
 31. The transgenic plant of claim 16, further defined as a monocotyledonous plant.
 32. The transgenic plant of claim 31, wherein said monocotyledonous plant is selected from the group consisting of wheat, maize, rye, rice, oat, barley, turfgrass, sorghum, millet and sugarcane.
 33. The transgenic plant of claim 16, further defined as a dicotyledonous plant.
 34. The transgenic plant of claim 33, wherein said dicotyledonous plant is selected from the group consisting of tobacco, tomato, potato, soybean, cotton, canola, alfalfa and sunflower.
 35. The transgenic plant of claim 34, wherein said dicotyledonous plant is a soybean plant.
 36. The transgenic plant of claim 16, further defined as a fertile R₀ transgenic plant.
 37. A method of expressing a selected protein in a transgenic plant comprising the steps of: (i) obtaining a construct comprising a selected coding region operably linked to a maize RS81 promoter; (ii) transforming a recipient plant cell with said construct; and (iii) regenerating a transgenic plant expressing said selected protein from said recipient plant cell.
 38. The method of claim 37, wherein said step of transforming comprises a method selected from the group consisting of microprojectile bombardment, PEG mediated transformation of protoplasts, electroporation, silicon carbide fiber mediated transformation, or Agrobacterium-mediated transformation.
 39. The method of claim 38, wherein said step of transforming comprises microprojectile bombardment.
 40. The method of claim 37, wherein said recipient plant cell is from a monocotyledonous plant.
 41. The method of claim 40, wherein said monocotyledonous plant is selected from the group consisting of wheat, maize, rye, rice, turfgrass, oat, barley, sorghum, millet, and sugarcane.
 42. The method of claim 41, wherein the monocotyledonous plant is a maize plant.
 43. The method of claim 37, wherein said recipient plant cell is from a dicotyledonous plant.
 44. The method of claim 43, wherein said dicotyledonous plant is selected from the group consisting of tobacco, tomato, potato, soybean, canola, sunflower, alfalfa and cotton.
 45. The method of claim 37, wherein said selected protein is selected from the group consisting of an insect resistance protein, a bacterial disease resistance protein, a fungal disease resistance protein, a viral disease resistance protein, a nematode disease resistance protein, a herbicide resistance protein, a protein affecting grain composition or quality, a nutrient utilization protein, a mycotoxin reduction protein, a male sterility protein, a selectable marker protein, a screenable marker protein, a negative selectable marker protein, a protein affecting plant agronomic characteristics, and an environment or stress resistance protein.
 46. A transgenic plant cell stably transformed with a selected DNA comprising a maize RS81 promoter.
 47. The transgenic plant cell of claim 46, wherein said maize RS81 promoter is isolatable from the nucleic acid sequence of SEQ ID NO:1.
 48. The transgenic plant cell of claim 46, wherein said maize RS81 promoter comprises from about 50 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 49. The transgenic plant cell of claim 46, wherein said maize RS81 promoter comprises from about 100 to about 2584 contiguous nucleotide s of the nucleic acid sequence of SEQ ID NO:1.
 50. The transgenic plant cell of claim 46, wherein said maize RS81 promoter comprises from about 200 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 51. The transgenic plant cell of claim 46, wherein said maize RS81 promoter comprises from about 400 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 52. The transgenic plant cell of claim 46, wherein said maize RS81 promoter comprises from about 750 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 53. The transgenic plant cell of claim 46, wherein said maize RS81 promoter comprises from about 1000 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 54. The transgenic plant cell of claim 46, wherein said maize RS81 promoter comprises from about 1500 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 55. The transgenic plant cell of claim 46, wherein said maize RS81 promoter comprises from about 2000 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1.
 56. The transgenic plant cell of claim 46, wherein said maize RS81 promoter comprises the nucleic acid sequence of SEQ ID NO:1.
 57. The transgenic plant cell of claim 46, wherein said selected DNA further comprises a selected coding region operably linked to said maize RS81 promoter.
 58. The transgenic plant cell of claim 57, wherein said selected coding region encodes an insect resistance protein, a bacterial disease resistance protein, a fungal disease resistance protein, a viral disease resistance protein, a nematode disease resistance protein, a herbicide resistance protein, a protein affecting grain composition or quality, a nutrient utilization protein, an environment or stress resistance protein, a mycotoxin reduction protein, a male sterility protein, a selectable marker protein, a screenable marker protein, a negative selectable marker protein, or a protein affecting plant agronomic characteristics.
 59. The transgenic plant cell of claim 58, wherein said selected protein is a selectable marker protein selected from the group consisting of phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase and glyophosate oxidoreductase.
 60. The transgenic plant cell of claim 46, wherein said selected DNA comprises plasmid DNA.
 61. The transgenic plant cell of claim 46, further defined as from a monocotyledonous plant.
 62. The transgenic plant cell of claim 61, wherein said monocotyledonous plant is selected from the group consisting of wheat, maize, rye, rice, oat, barley, turfgrass, sorghum, millet and sugarcane.
 63. The transgenic plant cell of claim 46, further defined as from a dicotyledonous plant.
 64. The transgenic plant cell of claim 63, wherein said dicotyledonous plant is selected from the group consisting of tobacco, tomato, potato, soybean, cotton, canola, alfalfa and sunflower.
 65. The transgenic plant cell of claim 64, wherein said dicotyledonous plant is a soybean plant.
 66. The method of claim 37, wherein said transgenic plant is fertile. 